New SPR based assays for plasma protein titer determination. - DiVA

Department of Physics, Chemistry and Biology

MASTER’S THESIS

New SPR based assays for plasma protein titer determination.

Johan Kärnhall

Performed at GE Healthcare Bio-Sciences AB

Linköping, February 2011

LITH-IFM-A-EX—11-2388--SE

The Department of Physics, Chemistry and Biology

Linköping University

SE-581 83 Linköping, Sweden

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Department of Physics, Chemistry and Biology

New SPR based assays for plasma protein titer determination.

Johan Kärnhall

Performed at GE Healthcare Bio-Sciences AB

Linköping, February 2011

Supervisors:

Åsa Frostell-Karlsson

Dr. Camilla Estmer Nilsson

Examiner:

Prof. Bo Liedberg

GE Healthcare Bio-Sciences AB

SE-750 15 Uppsala, Sweden

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Abstract Reliable analytical tools are important for time efficient and economical process development,

production and batch release of pharmaceuticals. Therapeutics recovered from human plasma,

called plasma protein products, involve a large pharmaceutical industry of plasma fractionation.

In plasma fractionation of human immunoglobulin G (hIgG) and albumin (HSA) recommended

analysis techniques are regulated by the European Pharmacopoeia and are including total protein

concentration assays and zone electrophoresis for protein composition and purity. These

techniques are robust, but more efficient techniques with higher resolution, specificity and less

hands-on time are available.

Surface plasmon resonance is an optical method to study biomolecular interactions label-free

in real time. This technology was used in this master thesis to set up assays using Biacore systems

for quantification of HSA and hIgG from all steps of chromatographic plasma fractionation as a

tool for process development and in-process control. The analyses have simplified mass balance

calculations to a high extent as they imply specific detection of the proteins compared with using

total protein detection. The assays have a low hands-on time and are very simple to perform and

the use of one master calibration curve during a full week decreases analysis time to a minimum.

Quick, in-process control quantification of one sample is easily obtained within <10 minutes. For

final QC of hIgG or for process development, an assay to quantify the distribution of the IgG

subclasses (1-4) was set up on Biacore and showed significantly lower hands-on time compared

with a commercial ELISA.

All assays showed reliable quantification and identification performed in unattended runs with

high precision, accuracy and sensitivity.

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Acknowledgement

I would like to thank:

My supervisors Åsa Frostell-Karlsson and Camilla Estmer Nilsson at GE Healthcare

Bio-Sciences for their great support and help throughout the project and for giving me

the opportunity to perform my master thesis project at GE Healthcare.

Members of the Protein Analysis R&D, Applications division for support and for

answering any Biacore-related questions.

Members of the BioProcessing section for their very friendly and supporting manner

during the three weeks of guidance and evaluation of the purification process and the

associated analyses. And for providing me process samples throughout the project.

Klara Pettersson, my opponent for carefully reading through this report and giving me

valuable feedback.

Bo Liedberg, for taking the time to be my examiner for this master‟s thesis project.

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Table of Contents

1 Introduction ............................................................................... 1

1.1. Background ....................................................................................................... 1

1.2. Aim ..................................................................................................................... 2

1.3. General approach ............................................................................................... 2

2 Theory ....................................................................................... 3

2.1. Plasma ................................................................................................................ 3

2.1.1. Plasma fractionation process ......................................................................................... 3

2.1.2. Immunoglobulin G ......................................................................................................... 6

2.1.3. Albumin ............................................................................................................................ 7

2.2. Protein characterization and quantification ...................................................... 8

2.2.1. Protein composition ....................................................................................................... 8

2.2.2. Molecular size distribution ............................................................................................. 8

2.2.3. Protein quantification ..................................................................................................... 8

2.2.4. International reference material .................................................................................... 9

2.2.5. Coefficient of Variation (CV) ...................................................................................... 10

2.3. Surface plasmon resonance biosensor technology .......................................... 11

2.3.1. Biacore system ............................................................................................................... 12

2.3.2. Sensor chip ..................................................................................................................... 13

2.3.3. Immobilization .............................................................................................................. 14

2.3.4. Concentration measurements ...................................................................................... 15

3 Materials and Methods ............................................................ 17

3.1. Materials .......................................................................................................... 17

3.1.1. Chemicals ....................................................................................................................... 17

3.1.2. Reagents .......................................................................................................................... 19

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3.1.3. Materials.......................................................................................................................... 20

3.2. Methods ........................................................................................................... 21

3.2.1. pH scouting .................................................................................................................... 21

3.2.2. Immobilization .............................................................................................................. 21

3.2.3. Regeneration .................................................................................................................. 22

3.2.4. Biacore concentration assay development ................................................................. 23

3.2.5. Activity and cross-reactivity experiment with capture antibodies .......................... 26

3.2.6. Value transfer from international reference material to calibrator ........................ 27

3.2.7. Biuret, total protein concentration assay ................................................................... 31

3.2.8. SDS-PAGE .................................................................................................................... 32

3.2.9. ELISA ............................................................................................................................. 34

4 Results ...................................................................................... 37

4.1. Total IgG concentration assay ........................................................................ 37

4.1.1. Evaluations of reagents for total IgG concentration assay ..................................... 37

4.1.2. Assay development total IgG concentration ............................................................. 37

4.1.3. International reference material calibration for IgG standard ................................ 41

4.1.4. Results total IgG assay on plasma-derived process samples .................................. 42

4.2. IgG subclass distribution assay ....................................................................... 46

4.2.1. Evaluations of reagents for IgG subclass distribution assay .................................. 46

4.2.2. Assay development IgG subclass distribution .......................................................... 52

4.2.3. International reference material calibration IgGSc-standard .................................. 55

4.2.4. Results IgG subclass distribution assay on plasma-derived samples ..................... 57

4.3. Albumin concentration assay .......................................................................... 64

4.3.1. Evaluations of reagents for albumin concentration assay ....................................... 64

4.3.2. Assay development albumin concentration .............................................................. 67

4.3.3. International reference material calibration for albumin standard ........................ 68

4.3.4. Results albumin assay on plasma-derived process samples .................................... 70

4.4. Albumin specificity assay ................................................................................ 74

4.4.1. Evaluations of reagents for albumin specificity assay .............................................. 74

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5 Discussion ................................................................................ 77

5.1. Total IgG concentration assay ........................................................................ 77

5.2. IgG subclass distribution assay ....................................................................... 77

5.3. Albumin concentration assay .......................................................................... 78

5.4. Biacore assays, performance and comparison ................................................ 78

5.4.1. Specificity ....................................................................................................................... 78

5.4.2. Sensitivity ........................................................................................................................ 79

5.4.3. Resolution ...................................................................................................................... 79

5.4.4. Robustness ..................................................................................................................... 80

5.4.5. Hands-on and analysis time ......................................................................................... 80

5.4.6. Consumables cost ......................................................................................................... 82

6 Recommendations ................................................................... 83

7 References ................................................................................ 85

Appendix A Regeneration scouting α-hIgG2........................................ 89

Appendix B Hands-on and analysis time .............................................. 91

Appendix C Protocol total IgG concentration assay ............................ 92

Appendix D Protocol IgG subclass distribution assay ......................... 94

Appendix E Protocol albumin concentration assay ............................. 97

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List of abbreviations CM5 Carboxymethylated Dextran 5 CV Coefficient of Variance EA Ethanolamine EDC 1-ethyl-3-dimethylaminopropyl-carbodiimide EDTA Ethylene diamintetra acetic acid HBS-EP+ 10 mM Hepes pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5 %

surfactant P20 IFC Integrated microfluidic cartridge IgG Immunoglobulin G IgGSc Immunoglobulin G subclass IVIG Intravenous immunoglobulin NHS N-hydroxysuccinimide P20 Surfactant P20 (Tween 20) RI Refractive Index RM Reference Material RU Resonance Unit SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SPR Surface Plasmon Resonance TM Target Material

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- INTRODUCTION -

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Chapter 1

1Introduction

1.1. Background

Today, plasma protein products recovered from human plasma is a major class of

therapeutics. A large pharmaceutical industry for fractionation of human plasma in the world

with over 70 factories exists [1]. During the development of fractionation processes, during the

execution of the process and for quality control (QC) there are high demands on good and

sensitive analytical tools. Analysis of plasma protein products is highly regulated for safety

reasons and current approved methods are presented in the European Pharmacopoeia by the

European Directorate for the Quality of Medicine and HealthCare [2].

Albumin has been used as a therapeutic for over 50 years and its main usage is for colloid

replacement and maintaining of blood volume at blood loss [3]. Intravenous Immunoglobulin G

has been used for over 25 years and mainly for replacement therapy in primary

immunodeficiency syndromes and for myeloma or chronic lymphatic leukaemia, but new areas of

use are emerging [4].

GE Healthcare Bio-Sciences AB in Uppsala, Sweden has a chromatographic plasma

fractionation process for the protein products coagulation factor VIII, factor IX, human serum

albumin and Immunoglobulin G from human blood plasma. The sensitivity, specificity, analysis-

and hands-on time of the available analysis methods were not satisfactory for the involved parties

who required new and better methods.

GE Healthcare‟s platform Biacore, which employs surface plasmon resonance biosensor

technology and is a highly sensitive label-free analysis tool for biomolecular interactions, was

chosen for the study.

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1.2. Aim

The first aim of this study was to perform a feasibility study to see which of the plasma

protein products that was possible to quantify satisfactory with a Biacore-assay, with focus on

albumin, Immunoglobulin G (IgG) and the relative distribution of Immunoglobulin G subclasses

(IgGSc) 1-4. The second aim was to develop the most viable assay as far as time allowed, in

addition the results and methods were to be compared with current alternative analyses.

1.3. General approach

Several antibody reagents will be tested and conditions optimized for the Biacore-system. The

extreme salt and pH conditions that occur from the purification steps could possibly interfere

with the interactions required for the analysis and these parameters needed investigation. Process

samples will be analysed with the new Biacore assay as it is developed as well as with current

methods as a comparison. The plasma fractionation process will be examined for insight into the

actual experimental situation.

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Chapter 2

2Theory

2.1. Plasma

2.1.1. Plasma fractionation process

Methods used for plasma fractionation has been developed since the 1946 with methods

varying from traditional cold ethanol fractionation with ethanol precipitation and centrifugation

as the major techniques to modern chromatographic processes [3]. The use of a chromatographic

process enables a larger variety of products to be extracted from the plasma other than traditional

albumin processes, it is also less damaging and generally gives a higher yield.

There are two types of human plasma differentiated by the means of collection. The major

type is plasma collected with plasmapheresis or apherisis where blood is filtered or continuously

centrifuged and the blood cells returned to the donor. The second type is plasma recovered

through double centrifugation of whole blood donations. Plasma from plasmapheresis

corresponds to 65 % and recovered plasma to 35 % of the total plasma fractionated in the world

today [1]. Both the plasmapheresis donations (category A plasma) and whole blood donations

(category B plasma) are to be frozen within 6 hours, if frozen within 24 hours of donation

(category C plasma) it can only be used in the production of immunoglobulin G and albumin [5].

The current process of interest is a chromatographic method using several steps of buffer

exchange chromatography, gel filtration chromatography, anion- and cation exchange

chromatography together with ultra- and diafiltration and numerous other steps. Ultrafiltration is

used to increase the concentration while diafiltration also replaces the buffer. An overview of the

process is displayed in Figure 2-1. The process is structured with factor VIII being the first

product to be separated, thereafter factor IX followed by albumin and finally IgG. This leads to

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four segments that can be called: factor VIII-trail, factor IX-trail, albumin-trail and finally IgG-

trail. The crude plasma has to be treated with heparin, which is a highly-sulphated

glycosaminoglycan acting as an anti-coagulant. All the products have to undergo virus

inactivation and sterile filtration in order to be safe to use as a pharmaceutical [1, 5]. Virus

inactivation is typically done by addition of solvent and detergent chemicals, such as tween-80,

TNBP, or triton X-100, or by pasteurisation and finally sterile filtration.

The chromatographic purification requires a variety of different buffers with different pH and

salt levels to elute the wanted proteins. Sodium Chloride (NaCl) levels vary between 0 and 500

mM and pH levels vary from pH 4.0 to pH 9.0. Together, this can yield quite extreme conditions

complicating the quantification methods.

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Figure 2-1: Process overview plasma fractionation by GE Healthcare The four sections are denoted Factor VIII-trail, Factor IX-trail, Albumin-trail and IgG-trail. Blue boxes represent chromatography steps and yellow boxes represent filtration steps. In this study only the Albumin- and IgG-trail were studied, each time starting from plasma following the black arrows. Samples were taken and analysed from the entire process, at least before and after every major chromatography and filtration step. For example the DEAE Sepharose FF step in the Albumin-trail was denoted “Alb DEAE” and the second ultrafiltration in the IgG-trail was denoted “IgG UF2”.

Plasma

Pre-treatment

Sepharose 4 FF

Q Sepharose HP

Chemical addition

Virus inactivation

SP Sepharose HP

Superose 12 pg

Formulation

Ultrafiltration

Sterile filtration

Filling

Lyophilisation

Severe heat treatment

DEAE Sepharose FF

Chemical addition

Virus inactivation

Heparin Sepharose FF

Q Sepharose FF

Ultra-diafiltration

Sterile filtration

Filling

Lyophilisation

Severe heat treatment

Ultrafiltration

Sephadex G-25 C

Euglobulin precipitation

Centrifugation

DEAE Sepharose FF

CM Sepharose FF

Ultrafiltration

Heat treatment

Centrifugation

Sephacryl S-200 HR

Ultra-diafiltration

Formulation

Sterile filtration

Ultrafiltration

Q Sepharose FF

Ultrafiltration

Chemical addition

Virus inactivation

CM Sepharose FF

Ultra-diafiltration

Formulation

Sterile filtration

Filling

Pasteurization

Filling

Factor VIII Albumin

Factor IX

IgG

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2.1.2. Immunoglobulin G

Immunoglobulins, also known as antibodies, are protein molecules part of the immune system

used to specifically identify and bind antigens leading to an immune response. Antibodies usually

bind the antigens tightly, sometimes not even leaving space for water molecules, by interactions

primarily formed by hydrogen bonds and electrostatic interactions. In the bloodstream the most

common class of immunoglobulins are immunoglobulin G class (IgG), which will hereby be

described more thoroughly. In a normal pool of plasma, the total IgG level is on average 8.5

mg/ml [6]. IgG is a globular, water-soluble protein with a molecular weight of approximately

150 000 Dalton (150 kDa). IgG is composed of two light chains consisting of two domains each

and two heavy chains consisting of four domains each, linked together with disulphide bonds, see

Figure 2-2 for a structural overview [7]. All domains possess the characteristic immunoglobulin

fold consisting of two sandwiched antiparallel β-sheets [8].

Immunoglobulins are glycoproteins containing of 82-96 % protein and 4-18 % carbohydrate

attached to the heavy chains [8]. Each IgG has two antigen binding sites located at the N-termini

of the light and heavy chains in the variable domains (Figure 2-2) [7]. The region on an antigen

recognized by the antibody is called the epitope; there can be several epitopes on one antigen

recognized by different antibodies.

Figure 2-2: Structural overview of Immunoglobulin G An illustration of Immunoglobulin G showing the heavy (red) and light (blue) chain and also the Fc and Fab regions [7].

The pharmaceutical product intravenous immunoglobulin (IVIG or IGIV in the US) has

many clinical uses but with potential risks and an inevitable limited supply due to its human

origin. The United States Food and Drug Administration (FDA) currently have six clinical

indications licensed for IVIG, they are: primary immunodeficiency disease, idiopathic

thrombocytopenic purpura, Kawasaki disease, B-cell chronic lymphocyticleukemia, HIV

infection, bone marrow transplantation [4]. In recent studies, it has also been found to work for

autoimmune diseases [9] and Alzheimer‟s Disease [10].

Antigen binding site

Fab

Fab

Fc

hinge region

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2.1.2.1. IgG subclasses

There are four different isotypes, or subclasses, of IgG named IgG1, IgG2, IgG3 and IgG4.

The distribution of these subtypes in the blood varies with individuals, depending mainly on age

and sex. The average distribution is as followed: IgG1 (58,9 %) > IgG2 (21,1 %) > IgG3 (4,3 %)

≈ IgG4 (4,7 %) [7]. These different IgG subclasses, illustrated in Figure 2-3, show differences in

structure where IgG3 is larger (170 kDa) than the others (146 kDa) with the main difference in

the hinge region, with 62 amino acids in IgG3 rather than 12 in the others. IgG3 is also more

susceptible to proteolytic enzymes and has a shorter biological half-time, 7 days compared to 21

days [7].

Figure 2-3: Immunoglobulin G subclasses Illustrations of the four IgG subclasses. The major visible differences are the hinge-region which is uniquely elongated in IgG3 and shorter in IgG4 [7].

2.1.3. Albumin

Albumin is the most abundant protein in the plasma and corresponds to approximately 60 %

of the total protein by mass. On average, in a normal pool of plasma, the albumin level is 34

mg/ml [6]. It is a very stable, highly water-soluble protein with a molecular weight of 66 500

Dalton (66.5 kDa) [11]. Albumin maintains the colloid osmotic pressure which ensures retaining

of water in the circulation. The protein is also a carrier for several hormones, enzymes, fatty-

acids, metal ions and medical products [3]. In the blood, albumin is generally composed with 0.5 -

1.5 moles fatty-acids per mole albumin [11]. The most frequent fatty-acids are: Oleic < 33 %,

Palmitic 25 %, and Linoleic < 20 % [11]. During purification, some of the fatty-acid composition

will be depleted and by special steps it can be completely removed yielding a fatty-acid free

albumin preparation [11].

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2.2. Protein characterization and quantification

2.2.1. Protein composition

The protein composition in a plasma sample is generally determined by sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins are separated on a gel by

electrophoresis, solely based on their molecular weight. By comparing the molecular mobility of

the samples with molecular markers, the protein composition and the purity may be concluded.

Other techniques for determination of protein composition are 2D gel electrophoresis (2DGE)

and capillary zone electrophoresis.

2.2.2. Molecular size distribution

Size exclusion chromatography, also called gel filtration chromatography, is used to determine

the molecular size distribution of the purified protein products. According to the European

Pharmacopoeia, for albumin at least 95 % of the total peak area has to be composed of monomer

or dimer and polymers and aggregates may not represent more than 5 % of the total peak area

[2]. For IgG the peaks of polymers and aggregates should not be more than 10 % of the total

peak area [2].

2.2.3. Protein quantification

Quantification of proteins is generally carried out with an assay based on analysis of a

calibrator of known concentration in several dilutions. In Biacore, there exists an alternative to

using a calibrator called Calibration Free Concentration Analysis (CFCA), more on this in section

2.3.4. The measured signal is used to construct a standard curve where standard points are fitted

with either a linear or non-linear mathematical fitting model. Samples with unknown

concentration with different dilutions is analysed and interpolated on the standard curve to give

the concentration. Preferably, a control sample with known concentration is also analysed and

the concentration interpolated on the standard curve is compared with the true concentration

[12].

Modern surface plasmon resonance based biosensor systems as well as nephelometric or

turbidimetric optical systems and ELISA use an antibody to recognize the targeted antigen in the

sample and these assays are called immunoassays or immunochemical assays. Other techniques

than immunoassays such as biuret-assay, Kjeldahl nitrogen-assay and absorbance spectroscopy

are less sensitive and not specific to a certain protein.

Quantification assays have a high demand on instrument and antibody reagents as well on

calibrators and controls. Immunoassays for human plasma protein measurements are highly

influenced by several factors that are not always met [13]. The nature of the antibody and antigen

is vital, with demand on highly specific antibodies and a homogenous invariable antigen. This is

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not always the case when analysing samples throughout a purification process as the antigen may

change as it becomes purer, for example the removal of fatty-acids bound to albumin (mentioned

in section 2.1.3) which may impact the interaction. Further, changes in salt levels as well as pH

might interfere with the antibody recognition in the immunoassay. Finally, the calibrator used has

to behave identically with the measured analyte in order to yield a comparable signal.

The leading techniques for protein quantification in clinical chemistry today are nephelometry

and turbidimetry [12]. The two methods are both immunochemical fluid phase optical sensors,

where nephelometry measures an increase in side-scattered light while turbidimetry measures a

decrease in light transmission. Calibrators or samples are injected into a reaction tube. Antibodies

against for example human IgG1 are added and antibody-antigen complexes are formed. After a

fixed time, the side-scattered light is recorded. Standard curves are constructed and sample

measures are interpolated and concentrations calculated [7].

There are several assays available for quantitative determination of IgG subclasses. The most

common ones are radial immunodiffusion (RID), nephelometry, turbidimetry and ELISA [7].

RID is performed in ready-to-use agar plates integrated with specific antibodies against the

IgGSc. Standards, controls and samples are added in holes in the agar. As the IgGSc migrates

into the agar and forms complexes with the integrated antibodies precipitation rings will emerge.

The diameter is proportional to the level of that specific IgG subclass. The method requires 48-

60 hours incubation time with a moderate hands-on time and no automation [7].

Nephelometry and turbidimetry are discussed above. The detection limit is in μg/ml range

with a fairly short analysis time and an automated system [7].

Enzyme-linked immunosorbent assay (ELISA) which was the method chosen to compare

with in this study is a well-known and widely used immunochemical method. The IgG subclasses

are captured by a coated anti-human IgG subclass-specific antibody. A secondary enzyme-linked

antibody is added and quantified by a coloured enzyme reaction upon addition of a substrate.

The ELISA method has a very low detection limit but demands a high hands-on time and a long

analysis time [7].

2.2.4. International reference material

In order to ensure the use of good and correct standards for quantification and to reduce the

observed variation of up to 50 – 100 % depending on the calibrator used, international reference

material has been introduced [13]. A variety of international reference materials has been used for

decades and has previously been produced by amongst others the World Health Organisation

(WHO), Community Bureau of References of the Commission of the European Communities

(BCR) and today by the Committee on Plasma Protein Standardisation of the International

Federation of Clinical Chemistry (IFCC) [13].

The latest recognised international reference material for plasma proteins is called ERM®-

DA470k/IFCC and is valid for twelve common plasma proteins: α2-macroglobulin, αl-acid

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glycoprotein (orosomucoid), αl-antitrypsin (αl-protease inhibitor), albumin, complement 3c,

complement 4, haptoglobin, immunoglobulin A, immunoglobulin G, immunoglobulin M,

transferrin and transthyretin (prealbumin) [14]. This type of reference material is called a certified

reference material (CRM) and is provided with a certificate of analysis with certified and traceable

values, accompanied with a value of uncertainty. CRMs are generally short on stock and are not

to be used on a daily basis [15].

Development and use of analytical tools requires large quantities of reference material and

with CRMs this would become quite costly. Instead it is recommended and practical to use other

reference materials or standards that are purchased or produced in-house to act as the calibrator

[15]. This calibrator is to be calibrated against the CRM using determined procedure and protocol

to transfer the value from the reference material to the target material [16-17].

2.2.5. Coefficient of Variation (CV)

The coefficient of variation (CV) is a normalized measure of reliability expressed in

percentage. It has the advantages to be a dimensionless number enabling the user to compare the

CV between different data sets without taking into consideration the mean value. When the mean

value is closer to zero the CV is very sensitive to small changes and are therefore not as useful.

CV is normally presented in percentage and with the number of data in the set as n. CV is

calculated with Equation 2-1 below.

Equation 2-1

Where σ = standard deviation and µ = mean.

100%

CV

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2.3. Surface plasmon resonance biosensor technology

Surface plasmon resonance (SPR) biosensor technology is a powerful tool in label-free

biomolecular interaction analysis used in drug discovery and proteomic research. Today, several

biosensor systems employing SPR technique exists on a growing market, the leader in SPR

biosensors is Biacore from GE Healthcare [18]. Also, other technologies for label-free

biomolecular interaction analysis are available, such as bio-layer interferometry (BLI) used in

ForteBio‟s instruments and quartz crystal microbalance used in Attana‟s and Q-sense‟s

instruments [19].

The application of surface plasmon resonance biosensors on biomolecules was first

demonstrated in 1983 [20]. When a beam of plane-polarized light passes through a prism with a

thin metal film it is totally internally reflected if the angle is above a certain critical angle of

incidence [21]. The reflected light is monitored and the intensity measured.

As the angle of incidence is changed the reflected light will decrease in intensity at a specific

angle showing a dip in reflected light. At this specific angle, surface plasmons in the metal film

are excited by the light inducing surface plasmon resonance (SPR) [21]. When the wave vector of

the incident light matches the wavelength of the surface plasmons, the free electrons in the metal

film resonate, hence the term surface plasmon resonance. The angle with the maximum loss of

intensity is called the SPR angle or resonance angle. This angle is dependent on the optical

properties of the media adjacent to the metal film.

Figure 2-4: Principle of SPR and schematic sensorgram Left: The principle of a SPR biosensor. Right: A schematic sensorgram showing the response upon association of analyte during injection and the dissociation post injection followed by regeneration.

1 0

Regeneration Analyte injection

phase Post-

injection

phase

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On one side is the glass prism with an unaltered high refractive index (RI) and on the other

side the measured medium with a low RI [21]. Molecules such as proteins are bound and thereby

increasing the mass, the refractive index is changed leading to a shift in the SPR angle [21]. Figure

2-4 illustrates the principles of SPR described here and also shows a schematic sensorgram. The

shift in angle is translated to resonance units (RU), 1 RU is equivalent to 10-4° corresponding to

about 1 pg/mm2 bound protein and is linear all the way to the upper limit of the dynamic range

[22].

The surface plasmon creates an electromagnetic field, called the evanescent field, which

propagates into the media [22]. Any change in mass, and thereby a change in RI, occurring

within this evanescent field is detected by the sensor [22]. The molecule immobilized on the

surface is called ligand and the molecule injected sample is called the analyte.

Since the response is dependant of the refractive index of the solution in the flow channel,

when a solution with a different RI is injected a bulk response will be visible. When the injection

is completed, the bulk response will disappear. This can for example be visible when using

different buffers and variable concentrations of salts, such as NaCl in the solutions. Extreme

levels of NaCl (very high or low) might also affect interactions in other ways, as many

interactions are governed by electrostatic attractions.

2.3.1. Biacore system

The Biacore system from GE Healthcare can monitor a biomolecular interaction in real-time

and label-free. The system consists of three main units, the SPR optics, the liquid handling

system and the sensor chip [21]. The sensor chip will be discussed in section 2.3.2 and the SPR

optics and the principle of SPR technology was brought up in section 2.3. The instrument used in

this study was Biacore T100 system and in some cases T200, see Figure 2-5. These instruments

are very similar but with a higher sensitivity in the T200. In this system the liquid handling system

consists of an IFC with four flow-cells, sample injection loops, highly accurate pumps and

pneumatic valves [23]. For different applications the flow-cells can be used independently or

serially as in Figure 2-6.

Figure 2-5: Biacore T100 instrument A Biacore T100 instrument that was used during this study.

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Figure 2-6: flow-cells. Illustration of the flow-cells in a Biacore T100 system [24]. The flow-cells are formed when the sensor chip is docked on the IFC (top). The four flow-cells can be used either serially (left) or independently (right).

2.3.2. Sensor chip

The sensor chip (see Figure 2-7) consists of a plastic cassette designed so the sensor chip is

easily moved and positioned automatically onto the integrated microfluidic cartridge (IFC) in the

instrument. The chip itself is composed of a thin glass covered with a 50nm gold film, coated

with a monolayer of hydroxyalkanethiol linkers [23]. There are several different sensor chips

available with different surface chemistry attached to the linkers; they are suitable for different

interactions, applications and immobilization techniques. The most common sensor chip, and the

chip used in this study, is the CM5; which has a carboxymethylated dextran matrix attached [22].

The dextran matrix is used as an anchor for the immobilization of ligands (see section 2.3.3).

There are more advantages by using a dextran matrix; firstly it enables the ligands to be

positioned in a three dimensional space increasing the number of interactions sensed by the

evanescent field and thereby increasing the binding response, secondly it enables the interaction

to proceed under conditions that mimics a fluidic and thirdly it minimises non-specific binding to

the gold surface [24].

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Figure 2-7: Series S sensor chip CM5 The carboxymethylated dextran matrix spans 100 nm into the flow-cell and is attached to the gold surface with a layer of linkers (grey).

2.3.3. Immobilization

There are several available coupling chemistries to immobilize proteins to the sensor chip

surface. To the carboxymethylated dextran matrix on CM5 sensor chip it is possible to do several

different coupling chemistries [25]. Amine coupling, the most used technique and the one used

in this study, will be further described below [26]. Carboxyl groups on the matrix forms covalent

bonds with primary amines on the ligand protein. This reaction does not occur spontaneously

and an activation of carboxyl groups into esters is necessary. This is done with a mixture of 1-

ethyl-3-dimethylaminopropyl-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as

illustrated in Figure 2-8.

First, EDC reacts with the carboxyl group forming a reactive intermediate. Second, the NHS

reacts and forms a NHS ester which is a good leaving group. Finally, as the protein is injected the

activated ester will spontaneously react with primary amines on the protein forming a covalent

bond. The final step is to inject an ethanolamine (EA) solution that reacts with the remaining of

the activated esters.

Figure 2-8: EDC NHS chemistry The chemical reaction during EDC/NHS immobilization. EDC reacts with the carboxyl group on the dextran matrix. This forms a reactive intermediate which reacts with NHS, leaving an ester. The NHS ester is a good leaving group, reacting with a primary amine on the ligand, forming a covalent bond.

Carboxyl group / Ligand

Dextran

Linker layer

Gold film

Glass

- THEORY -

- 15 -

During the immobilization, in order to attract the ligand to the surface to ensure the reaction

to occur to a satisfactory extent it has to be attracted by electrostatic forces in something called

pre-concentration. By dissolving the ligand in a buffer with a pH below the pI of the protein, this

will result in the protein having a net positive charge and it will be attracted to the slightly

negatively charged dextran matrix.

The optimum pH can be determined by a pH scouting experiment described in section 3.2.1.

The properties that can be modified in order to vary the level of final immobilized ligand to the

desired level are concentration, pH of buffer, flow-rate and contact time.

2.3.4. Concentration measurements

The SPR technology can be used for concentration measurements in a robust, accurate,

precise and specific manner [27]. In several recent studies, SPR biosensors have been used for

quantification, for example quantification of bovine IgG in milk (2010) [28], estriol metabolites in

liquid media (2009) [29] and yessotoxin from marine dinoflagellates (2008) [30]. In general, three

methods for quantification exist. A traditional method using the relative response after injection

[23], a method using the binding rate (RU/s) [31] and the most recent calibration free

concentration analysis (CFCA) using two different flow-rates [32].

- MATERIALS AND METHODS -

- 17 -

Chapter 3

3Materials and Methods

3.1. Materials

3.1.1. Chemicals

Chemical Cat. No. Supplier

Milli-Q filtered H2O Millipore

HBS-EP+ 10X BR-1006-69 GE Healthcare

MgCl2 M2670 Sigma-Aldrich

50 mM NaOH BR-1003-58 GE Healthcare

3M MgCl2 BR-1008-39 (capture kit) GE Healthcare

Glycine pH 2.0 BR-1003-55 GE Healthcare

Amine coupling

Ethanol amine BR-1000-50 GE Healthcare

EDC BR-1000-50 GE Healthcare

NHS BR-1000-50 GE Healthcare

Immobilization pH scouting

Acetate pH 4.0 BR-1003-49 GE Healthcare




- CHAPTER 3 -

- 18 -

Regeneration Scouting

Glycine pH 1.5 Regeneration scouting kit GE Healthcare




SDS 0.5% BR-1005-56 GE Healthcare

NaCl 5M BR-1005-56 GE Healthcare

MgCl2 4M BR-1005-56 GE Healthcare

NaOH 200mM BR-1005-56 GE Healthcare

Ethylene Glycol BR-1005-56 GE Healthcare

SDS-PAGE NuPAGE Novex 4-12% Bis-Tris Gel, 1.9 mm, 12 well NP0322BOX Invitrogen

Precision Plus Protein Dual Color Standard 161-0374 Bio-Rad Laboratories AB

NuPAGE LDS sample buffer 4X NP0007 Invitrogen

β-Mercapthoethanol M6250 Sigma-Aldrich

NuPAGE MOPS SDS Running buffer 20X NP0001 Invitrogen

GelCode Blue Stain Reagent #24592 Thermo Scientific

ELISA

Peliclass human IgG subclass kit M1551 Sanquin

Chromatography media

Ion exchanger media ( GE Healthcare)

Matrix: Highly cross-linked agarose, 6%

Particle size: average 90 μm (45 – 165 μm)

Q Sepharose™ FF Quaternary ammonium strong anion exchanger

Cat. No. 17-0510-05

DEAE Sepharose™ FF Diethylaminoethyl weak anion exchanger

Cat. No. 17-0709-05

CM Sepharose™ FF Carboxymethyl weak cation exchanger

Cat. No. 17-0719-05

Gel filtration media (GE Healthcare)

Sepharose 4 Fast Flow Highly cross-linked 4% agarose

Cat. No. 17-0149-01 Particle size: 45 – 165 µm

Fractionation range: 6 × 104 – 3 × 107 Da

Sephacryl S-200 HR Spherical allyl dextran and N, N’-methylenebisacrylamide

Cat. No. 17-0584-10 Particle size: 50 µm

Fractionation range: 5 × 103 – 2.5 × 105 Da

Sephadex G-25 Cross-linked dextran

Cat. No. 17-0034-01 Particle size: 75 – 510 µm

Fractionation range: 1 × 103 – 5 × 103 Da


- 19 -

3.1.2. Reagents

Name Denotation Supplier / Cat. No.

human IgG hIgG Sigma / I4506

human IgG1 κ (myeloma) hIgG1 Millipore / AG502




Peliclass human IgG subclass standard IgGSc-standard Sanquin / M1551

Peliclass human IgG subclass control IgGSc-control Sanquin / M1551

HSA "Essentially fatty acid free" HSAa Sigma / A-3782

HSA "Fraction V" HSAb Sigma / A-1653

HSA “internally purified” HSAc GE Healthcare / internal

BSA BSA Sigma / P9418

HSA and gamma-globulins Sigma / P8119

International RM - ERM-DA470k/IFCC ERM-DA470k Sigma / ERMDA470KIFCC-1VL Antibodies

Species +

Specificity Denotation Clone Isotype Supplier / Cat. No.

human IgG (Fc) α-hIgG GE Healthcare / BR-1008-39

human IgG1 α-hIgG1poly sheep (poly) The binding site / AU006

human IgG1 (Fc) α-hIgG1a HP6091 mouse IgG2a The binding site / MC003

human IgG1 (Fc) α-hIgG1b HP6069 mouse IgG1 Invitrogen / MH1013

human IgG1 (Fc) α-hIgG1 HP6070 mouse IgG1 Invitrogen / MH1015

human IgG2 (Fab) α-hIgG2 HP6014 mouse IgG1 The binding site / MC005

human IgG3 (Fab2) α-hIgG3 HP6050 mouse IgG1 The binding site / MC006

human IgG4 (pFc) α-hIgG4 HP6025 mouse IgG1 The binding site / MC009

mouse IgG (Fc) α-mIgG Rabbit (poly) GE Healthcare / BR-100838

HSA α-HSApoly Rabbit (poly) GE Healthcare / internal

HSA α-HSAmab mouse IgG1 Abcam / Ab399

BSA α-BSAa 2A3E6 mouse IgG1 Santa Cruz Biotech / sc-32816

BSA α-BSAb 0.N.32 mouse IgG1 Santa Cruz Biotech / sc-70445

BSA α-BSAc BGN/D1 mouse IgG1 Santa Cruz Biotech / sc-80704

- CHAPTER 3 -

- 20 -

3.1.3. Materials

Material Cat. No. Supplier

Microplate 96 well

Microplate cover-foil 96 well

Microplate flat bottom 96 well

Pipette and pipette tips, 10-100ul Eppendorf



Finnpipette, 5mL Labsystems

Finntips, 5mL 940 20 50 Thermo Scientific

Pipette Multi channel, 30-300ul Eppendorf

Pipette Multi channel automatic, 10-200ul Eppendorf

Pipette Multi channel automatic, 100-1000ul Eppendorf

Series S Sensor chip CM5 BR-1006-68 GE Healthcare

Plastic vials, ø 7mm BR-1002-12 GE Healthcare

Glass vials, ø 16mm BR-1002-09 GE Healthcare

Rubber cap, type 3 (for ø 7mm) BR-1005-02 GE Healthcare

Rubber cap, type 2 (for ø 16mm) BR-1004-11 GE Healthcare

Instrument Software Supplier

Biacore T100 Control software v2.0.3, Evaluation software v2.0.3 GE Healthcare

Biacore T200 Control software v1.0, Evaluation software v1.0 GE Healthcare

Milli-Q Advantage A10 Millipore

Electrophoresis power supply – EPS 301 GE Healthcare

miniVE – Vertical electrophoresis system GE Healthcare

ImageScanner III Labscan 6.0 GE Healthcare

ImmageQuant TL 6.0 GE Healthcare

SPECTRA Max PLUS 384 SoftMax Pro v5.4 Molecular Devices

Microplate-shaker

ÄKTA pilot Unicorn v5.11 GE Healthcare


- 21 -

3.2. Methods

If nothing else is stated, all Biacore-experiments were performed at 25°C with HBS-EP+ as

sample and running buffer. For longer (>12 hours) experiments the sample compartment

temperature was decreased to 10°C from 25°C, but the analysis temperature remained unaltered.

3.2.1. pH scouting

In order to determine the optimal pH for pre-concentrating the ligand to the matrix during

immobilization, as described in section 2.3.3, a pH scouting was performed. The ligand was

diluted to 20 μg/ml in buffers with different pH and injected during 2.5 minutes over an

unmodified sensor chip. After each injection the surface was regenerated with 50 mM NaOH to

ensure no ligand remains non-specifically bound to the surface. The most neutral pH was

injected first followed by more acidic injections. The aim was to obtain a sufficiently high

increase of response but with the most neutral pH possible in order to maintain the native state

of the ligand. The buffers used were 10 mM sodium acetate with pH ranging from 4.0 to 5.5, 10

mM maleate pH 6.0 to 6.5 and 10 mM phosphate pH 7.0. An example of a pH scouting can be

seen in Figure 4-8.

3.2.2. Immobilization

Immobilization of ligands to the sensor chip surface was performed with amine coupling

chemistry, as described in section 2.3.3. Chemicals from amine coupling kit (GE Healthcare)

were utilised. The surface was activated with a 7 minute injection of 1:1 mixture of EDC and

NHS. The ligand injection was optimized for each antibody and specified under each result

section; typically a 7 minute injection of 20 μg/ml antibody diluted in pre-concentration buffer

was used. The surface was deactivated with a 7 minute injection of ethanol amine (EA). An

example of an immobilization sensorgram is displayed in Figure 3-1.

Figure 3-1: Typical immobilization sensorgram

0

10000

20000

30000

0 400 800 1200 1600

Re

spo

nse

(RU

) .

Time (s)

EA Ligand ~10000 RU EDC/NHS

- CHAPTER 3 -

- 22 -

3.2.3. Regeneration

For experiments when the affinity of the interaction is high, and the analyte does not

dissociate by itself it is required to regenerate the surface between cycles. This is generally the

case for concentration analysis with high affinity antibodies and high responses. The principle of

regeneration is that the interactions between the analyte and the ligand are broken at the same

time as the analyte may be partly denatured whilst the ligand maintains its activity. Therefore, for

an easier regeneration the less stable protein should be the analyte.

Different results that might occur during regeneration are illustrated in

Figure 3-2. A and B show optimal and acceptable regeneration when the analyte response and

the baseline remains the same. C and D illustrate incomplete regeneration. The last two show

irreversible changes of the ligand due to the regeneration, E has a loss of ligand activity and in F

the ligand is lost from the surface.

Figure 3-2: Illustration of regeneration results Common regeneration results are illustrated. A and B show good regeneration. C and D illustrate incomplete regeneration. E and F indicate an irreversible change on the ligand due to regeneration.

3.2.3.1. Regeneration scouting

The protocol from the regeneration scouting kit was followed. A freshly immobilized and

previously unused surface was used for each regeneration solution tested. An analyte with a high

concentration was injected and the binding response and baseline was compared to the initial

cycle. The mildest condition for each solution was used first with a successively tougher

Analyte response

Baseline

A Optimal regeneration.

C Incomplete regeneration. Accumulation of analyte and loss of capacity. .

E Loss of ligand activity. Irreversible change.

B Acceptable regeneration.

D Incomplete regeneration. Accumulation of analyte.

F Loss of ligand. Irreversible change.


- 23 -

condition following. For each condition the analyte was injected and regenerated four to five

times.

The conditions are met if the response is recovered to preferably 70 % from the first cycle and

the baseline is similar to the first cycle, a small constant decrease in baseline may be acceptable as

long as the analyte response is repeatable. The condition that gives the best regeneration is

verified by 20 or more cycles with the same condition. Further, the injection time of regeneration

solution might be increased or decreased in order to give a better regeneration.

The tested regeneration solutions were:

10 mM Glycine-HCl, pH 3.0 to 1.5

Ethylene glycol, 50% to 100%

Sodium hydroxide (NaOH), 1 mM to 75 mM

Magnesium chloride (MgCl2), 1 M to 4 M

Sodium chloride (NaCl), 0.5 M to 5 M

Sodium dodecyl sulphate (SDS), 0.02 % to 0.5 %

3.2.4. Biacore concentration assay development

There were three different methods of concentration determination in Biacore to choose

from. First, the traditional method where the relative response of the calibrator was plotted

against the concentration [23]. Second, a method where the binding rate (RU/s) of the calibrator

was plotted against the concentration [31]. Third, a calibration free concentration analysis

(CFCA) where a calibrator was not needed by using different flow-rates [32-33]. With the plasma

and process samples that were analysed, the traditional relative response method was chosen due

to large bulk responses and some non-specific binding during injection of non-purified samples

interfering with the other methods.

The Biacore concentration assays that were developed in this study had a number of

parameters that were optimized and thus leading to the assays presented in section 4.1.2 for total

IgG, section 4.2.2 for IgG subclass distribution and section 4.3.2 for albumin. These parameters

and the criterions to determine them will be discussed here.

Biacore concentration assay parameters:

Ligand antibody

o Choice of antibody

The desired characteristics for an antibody to be used in a concentration assay

was that it binds the analyte specific and with a high affinity when immobilized

on the sensor chip. Antibodies were also necessary to be able to regenerate

under known conditions without losing activity. Preferably commercially

available monoclonal antibodies were chosen.

- CHAPTER 3 -

- 24 -

o Immobilization level: Buffer, injection time, flow-rate, concentration

The aimed immobilization level for concentration assays is generally around

10000 RU. A high immobilization level is necessary in order to have mass-

transport limited interaction as discussed below. To reach a certain level the

pH of the pre-concentration buffer had to be determined by a pH scouting

(section 3.2.1). Also the injection time was evaluated to obtain desired level.

Finally the concentration of the antibody diluted into the pre-concentration

buffer was studied to determine a suitable concentration. Several antibodies

were delivered in sodium-azide preservative and Tris-buffer and had to be

diluted enough to avoid interfering with the immobilization. As these

compounds contain a primary amine they would otherwise be immobilized.

The flow-rate decreased in order to reduce consumption of reagents.

Concentration assay

o Buffer

HBS-EP+ has in several previous studies been shown as an appropriate buffer

for real-time interaction studies and was found to work well also for these

assays.

o Choice of reagent

The reagent used as standard needs to interact with the antibody in an identical

manner as the sample. The reagent should preferably be commercially

available. Users of the assays can utilise their own standards as long as it is

calibrated against the international reference material.

o Concentration range, injection time and flow-rate

Injection time and concentrations were varied to obtain an assay where the

lowest point in the standard curve gave high enough response while

maintaining sufficient sensitivity. At the same time the assays were designed to

be as rapid as possible. The time could readily be shortened as the sensitivity

was not the main focus, since the samples generally had high concentrations.

The highest point in the standard curve was chosen so the interaction would

be mass-transport limited and thus having a linear increase of response during

the injection and avoiding the antibodies to approach steady-state [31, 33].

This lead to linear standard curves without a plateau, consequently giving a

higher resolution and precision. The dilutions of the standard were typically

done by six serial 2- or 2.5-fold dilutions.

Even though the flow-rate might affect the response slightly this parameter

was normally only set to reduce sample consumption.


- 25 -

o Regeneration: Conditions, injection time, flow-rate

If not previously known, the regeneration conditions were found by

regeneration scouting (section 3.2.3.1). The flow-rate was typically set slightly

higher than the flow-rate for the analyte injection. The injection time of

regeneration solution was also kept as short as possible to have a short analysis

time but with a complete regeneration. A so called pre-dip was used to avoid

dilution of the regeneration solution with running buffer during analysis of

many samples.

An example of a sensorgram from the injection of a standard curve is shown in Figure 3-3.

Some of the parameters discussed above are also illustrated in the figure. The relative response

was found by subtracting the baseline response, before injection, from the response after

injection as illustrated. Also seen in the figure is the short sample injection time with almost

completely constant RU/s. The delay between end of injection and regeneration was limited by

IFC washing in the instrument.

Figure 3-3: Example of sensorgrams from injection of standard curve. Illustrating standard curves from six concentrations of standard. In the example the injection and regeneration time are illustrated with arrows.

x x

[standard] (µg/ml)

0

1000

2000

3000

0 20 40 60 80 100 120 140 160

Time (s)

x

x

x

x x

50

20

3.2

8

1.3 0.5

Injection Regeneration

Response (RU)

x

- CHAPTER 3 -

- 26 -

3.2.5. Activity and cross-reactivity experiment with capture antibodies

The set-up for an experiment with a capture antibody was used to ensure the mildest possible

treatment of the antibody by relieving it from the stress of being immobilized by acidic

conditions during covalent coupling. The set-up is also preferred if the regeneration conditions

for the antibody are not yet known.

It is possible to get false negative results if the immobilized ligand binds to the binding

domain of the capture antibody making it unable to bind its antigen. To eliminate false positive

results it is essential to also inject the analyte without the capture antibody to ensure it does not

interact with the ligand alone.

The set-up is illustrated in Figure 3-4 supported with a schematic sensorgram. As the capture

antibody is injected there is an increase in response. If there is another increase in response as the

analyte is injected it is considered a positive interaction. This is followed by regeneration of the

surface and a second capture antibody can be injected.

Figure 3-4: Set-up of method with capture antibody The first antibody (blue) represents the ligand immobilized to the dextran matrix on the sensor chip. The capture antibody (red) is injected giving a response seen in the sensorgram. As the analyte (green) is injected it gives a response in the sensorgram if the interaction is positive.

Inject capture Ab

Inject sample Positive!

Regeneration

Immobilized ligand Capture antibody Analyte

Re

spo

nse

(RU

)

Time (s)


- 27 -

3.2.6. Value transfer from international reference material to calibrator

A protocol developed by the International Federation of Clinical Chemistry and Laboratory

Medicine (IFCC) to transfer plasma protein concentration values from an reference material

(RM), here ERM-DA470k/IFCC [14], to an internal calibrator, called target material (TM), was

followed [16-17]. The procedure will be described here and deviations from the protocol will be

accentuated. The value transfer was performed after the assays were completed. While the

procedure is described here the results for each of the three assays are presented in their

respective result section (4.1.3 for total IgG, 4.2.3 for IgG subclass distribution and 4.3.3 for

albumin).

According to the protocol, the measurements were to be performed three times a day on four

consecutive days, but due to time constraints the measurements were only performed once a day

over three days but with duplicate measurements for both calibration curve and samples. For

each day, new dilutions were made. Both the calibration curve and the sample consisted of six

dilutions each. This yielded in 36 determinations each (6 dilutions * 2 replicates * 3 days = 36) for

the RM and the TM. An additional special dilution of RM was used as control sample giving

another 6 determinations (2 replicates * 3 days = 6).

The RM was reconstituted according to the product sheet:

The vial was thawed in room temperature for one hour.

The vial was tapped gently to ensure all material settled on the bottom.

Removing the screw cap.

The vial together with rubber stopper was weighed in gram with four decimals.

1 mL of water was added, new weight recorded to acquire the water mass.

The concentration after constitution was calculated with Equation 3-1 below.

After one hour, the vial was inverted gently five times during one hour.

Vail stored in room temperature overnight.

Equation 3-1

Where is the certified concentration and the actual concentration after

reconstitution.

Six dilutions of the reconstituted RM served as standards for the calibration curve. The

concentrations were evenly distributed over the measuring range of the assay. To minimize the

sources of errors all volumes dispensed were controlled by weighing and the actual dilutions with

four decimals were calculated. The densities of all liquids were approximated to 1.

water

R

water

RR

m

C

m

mCC

0000.1''

'

RC RC

- CHAPTER 3 -

- 28 -

An example of the dilution scheme for human IgG is presented in Table 3-1 below. In order

to get suitable volumes of RM and dilution buffer in the scheme, a predilution of the RM was

performed. Additionally, to avoid pipetting small volumes, Std.4, Std.5 and Std.6 were prepared

from Std.2, Std.3 and Std.5 respectively.

The relative concentration in percentage was calculated by Equation 3-2 and these

concentrations represent the values on the x-axis in the calibration curve. Another excel-

spreadsheet equivalent to that in Table 3-1 was filled in with actual masses from pipetting where

actual dilutions and relative concentrations were calculated. Hence, these were the values entered

into the method as concentration in percentage.

Equation 3-2

Where is the mass of reference material, the mass of dilution buffer,

predilF is the predilution factor and Std.2 is the relative concentration of Standard 2.

Human IgG - Intended predilution of the Reference Material

Dilution buffer, (g) 0,9500

Reference Material, (g) 0,0500

Predilution factor, FPredil 0,050000 RM reconst. conc. (CR)

Human IgG - Intended dilutions of the Reference Material 9259,82 μg/ml

Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6 Control

Dilution buffer, (g) 0,8000 1,2000 1,3000 0,4000 0,7000 0,6000 1,1000

Reference Material, (g) 0,1000 0,1000 0,0750 0,3000 0,3000 0,1000 0,0500

of predil of predil of predil of Std. 2 of Std. 3 of Std. 5 of predil

Total mass, (g) 0,9000 1,3000 1,3750 0,7000 1,0000 0,7000 1,1500

Dilution factor 0,1111 0,0769 0,0545 0,4286 0,3000 0,1429 0,0435

Predilution factor 0,050000 0,050000 0,050000 0,003846 0,002727 0,000818 0,050000

Relative concentration, (%) 0,5556 0,3846 0,2727 0,1648 0,0818 0,0117 0,2174

Concentration, (μg/ml) 51,44 35,61 25,25 15,26 7,58 1,08 20,13

Aimed target conc. (μg/ml) 50,00 35,00 25,00 15,00 7,50 1,00 20,00

Table 3-1: Example of intended dilution scheme of reference material for value transfer Predilutions for Std.4, Std.5 and Std.6 were calculated from the dilution of the standard it was prepared from. All weights were recorded in grams with four decimals and predilution factors with six decimals. Volumes were chosen to give a concentration in μg/ml (calculated from CR and the relative concentration) close to the aimed target concentration, which was based on the concentration range of the assay. This dilution scheme was followed and an equivalent excel-spread sheet was filled in with actual masses where actual dilutions and actual relative concentrations were calculated.

RM DilM

(when Std.4 is prepared

from Std.2)

or

DilR

Rpredil

MM

MFStdconcrel

1002..

DilR

R

MM

MStdStdconcrel

2.4..


- 29 -

The target material was also diluted in six dilutions. As these six dilutions were samples they

were aimed to all fall within the calibration curve generated. A predilution was performed to a

concentration in the upper quarter of the standard curve. The prediluted TM was added in

decreasing volumes to correspondingly increasing volumes of dilution buffer yielding in a

constant total volume, with all weights recorded. An example of dilution scheme of target

material for IgG is shown in Table 3-2. The dilution factor denoted FT2 was calculated by

Equation 3-3 and was used as the x-value in the upcoming plot and calculations. The actual

masses were weighed and entered in an excel-spreadsheet giving the actual values of FT2.

Equation 3-3

Where is the mass of prediluted target material and the mass of dilution buffer.

Human IgG - intended predilution of the Target Material

Dilution buffer, (g) 2,5000

Target Material, (g) 0,0400

Predilution factor, FT1 0,015748

TM estimated conc.

Human IgG - intended dilutions of the Target Material 2000,00 μg/ml

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Dilution buffer, (g) 0,0000 0,0500 0,1000 0,2000 0,3000 0,3750

Target Material, (g) 0,5000 0,4500 0,4000 0,3000 0,2000 0,1250

Dilution factor, FT2 1,0000 0,9000 0,8000 0,6000 0,4000 0,2500

Predilution factor, FT1 0,015748 0,015748 0,015748 0,015748 0,015748 0,015748

Relative concentration, (%) 1,5748 1,4173 1,2598 0,9449 0,6299 0,3937

Concentration, (μg/ml) 31,50 28,35 25,20 18,90 12,60 7,87

Table 3-2: Example of intended dilution scheme of target material for value transfer The highlighted values are dilution factor FT2. All weights were recorded in grams with four decimals. The actual values from the corresponding excel-spreadsheet will be used as x-values in the upcoming plot and calculations.

When all dilutions were made, the assay was executed with one set of standards, duplicates of

all samples, a duplicate of control sample and finally another set of standards. The outcome was a

standard curve similar to the illustration in Figure 3-5, with relative concentration in percentage

versus response in RU. In the evaluation software, the responses of the samples were

interpolated on the standard curve giving a relative concentration in percentage.

Figure 3-5: Schematic standard curve value transfer The standard curve with relative concentration in percentage on the x-axis and response in RU on the y-axis.

DilT

TT

MM

MF

2

TM DilM

Response, (RU)

Relative concentration, (%)

- CHAPTER 3 -

- 30 -

The average concentration for each sample was transformed with Equation 3-4 to become a

y-value, or relative concentration factor FR, that was comparable with the x-values, FT2. The

control samples were compared to expected relative concentrations to give an indication of assay

performance. This was done by dividing the interpolated relative concentration with the expected

relative concentration; a value of 1.0 equals a control with 100 % compared to expected.

Equation 3-4

Where FR is the relative concentration factor for sample i and FT1 is the predilution factor.

The dilution factor FT2 was then plotted against the measured relative concentration factor FR

for all six samples. This gave a plot similar to Figure 3-6. A linear regression ( ) was

performed and if a zero intercept was within the confidence interval a new regression was

performed with the intercept set to zero ( ). A zero intercept means that there was no

matrix effects in the assay, i.e. the buffer conditions were the same for TM and RM after

dilutions.

Figure 3-6: schematic plot of FT2 versus FR for value transfer Linear regression with intercept set to zero for dilution factors plotted against relative concentration factors. The slope is equal to the ration of target material and reference material concentrations as shown in Equation 3-5.

According to Blirup-Jensen et al [16], derivation not shown here, the slope of the line is equal

to the ratio between the target and reference material concentrations as in Equation 3-5.

Accordingly, the actual target material concentration CT was calculated by the right side of

Equation 3-5.

Equation 3-5

Where CT is the actual target material concentration, CR the reconstituted reference material concentration calculated in Equation 2-1 and β the slope from the linear regression.

1001

)(

T

RF

isampleaverageiF

XY

0

RT

R

T CCC

C

y-value: relative concentration factor, FR

x-value: Dilution factor, FT2


- 31 -

A transfer factor (TF) was calculated with Equation 3-6 for transformation of results done

prior to this reference calibration. Previous results were multiplied with the TF giving the correct

concentrations.

Equation 3-6

Where CT is the actual target material concentration after value transfer and is

the previously known concentration of the target material (if applicable).

This whole procedure was performed on three consecutive days giving three linear regressions

with one slope each, giving three values on the actual target material concentration CT from

which the average target material concentration was calculated. The results for each assay are

presented in section 4.1.3 for total IgG, 4.2.3 for IgG subclass distribution and 4.3.3 for albumin.

3.2.7. Biuret, total protein concentration assay

The total protein concentration was determined with the biuret assay as described in the

European Pharmacopoeia 2.55.3 [2]. The assay involves a reaction in alkali solution between

cupric ions and peptide bonds to form a complex with absorbance at 546 nm. Preparation of the

biuret solution is described below. A protein standard containing both HSA and hIgG was used

(80mg/ml, Sigma). Two different standard curves and protocols were used depending on the

estimated samples concentration; see table x. The standard curves included 5 points in 2 times

serial dilutions. The larger volume of the standards/samples for the low calibration curve was due

to that the absorbance should be between OD 0.1-1 to be optimal in measurement.

Volume Standard

5-80 mg/ml Standard 0.5-6 mg/ml

Standard 10µl 100µl

Sample 10µl 100µl

Biuret solution 200µl 100µl

Table 3-3: Volumes used for two standard curves.

Standards and samples were added in duplicates into a flat bottom 96-well microplate. Biuret

solution was added and the microplate was incubated for 30 minutes on a shaker. The

absorbance was measured in a plate reader at 541 nm and the concentration calculated by

constructing a linear calibration curve.

Preparations of biuret solution:

3.0 g CuSO4x5 H20 + 9.0 g C4H4KNaO6x4 H20 + 5.0 g KI

Add 800 mL milli-Q water, stir until dissolved

Add 100 mL 6.0 M NaOH

Fill up to 1000 mL with milli-Q water

old

T

T

C

CTF

old

TC

- CHAPTER 3 -

- 32 -

3.2.8. SDS-PAGE

In order to calculate the specific IgG or albumin concentration with traditional methods, the

purity in percentage was estimated by SDS-PAGE and then multiplied with the total protein

concentration from biuret.

The samples were diluted with water according to Table 3-4 to get suitable amounts of protein

on the gel. To reduce the proteins prior to loading the samples were mixed with sample loading

buffer (10 µl sample + 10 μl 4X NuPAGE sample buffer with 20% β-mercapthoethanol) and

heated at 70°C for 10 minutes.

Sample concentration (µg/µl) Dilution

1-2 2x

2-9 5x

10-19 10x

20-70 50x

70-90 100x

>100 200x

Table 3-4: Sample dilutions for SDS-PAGE Simplified dilution scheme of samples in order to load an appropriate amount of protein onto the gel to avoid over-load or not having enough protein.

The gel was docked to the electrophoresis system and running buffer (NuPAGE MOPS SDS

running buffer, Invitrogen) was added. 5 μl of molecular weight marker (Precision Plus Protein

Dual Color Standard, Bio-Rad Laboratories AB) was added to the first lane and 10 μl of sample

mixture to all other lanes. The gel was run for 10 minutes with 60 V to gather the protein bands

below the wells followed by 70 minutes with 150 V. The protein bands were stained using

GelCode blue staining kit (Thermo Fisher Scientific) over night while shaking, then destained in

water for another 24 hours.

Dyed gels were scanned on an ImageScanner III using Labscan 6.0 (GE Healthcare) and

analysed in ImageQuant TL 6.0 (GE Healthcare). The peaks on each lane were identified and

cut-offs determined. The known proteins in the samples, such as IgG, albumin and transferrin,

were recognized. An example gel is shown below in Figure 3-7 with the “pixelogram” analysis of

lane 10 in Figure 3-8. From the area under the curves the relative quantity of that specific protein

was calculated in relation to the total curve area in the lane. As seen in Figure 3-8 the relative

quantity of each protein was an estimation and therefore not highly accurate. The relative

quantity was also interpreted as the purity of the proteins in the sample.


- 33 -

Figure 3-7 (left): Scanned picture of a SDS-PAGE gel Lane 1 contains the molecular weight marker and lane 2 to 12 samples from different steps in the plasma fractionation process.

Figure 3-8 (right): Analysis of lane 10 from SDS-PAGE Example showing the analysis of lane 10 from the gel in Figure 3-7. Peak 5 at 70 kDa was believed to corresponds to transferrin, peak 6 and 7 at 62 and 57 kDa to albumin and finally peak 9 and 12 at 50 and 21 kDa to IgG. The relative quantity, thereby also the purity, was calculated to 51 % for albumin and 18 % for IgG in this sample.

As IgG consists of several chains linked with disulphide bonds more than one band will

appear on a reduced gel as illustrated in Figure 3-9. If detected they were added for the full IgG

composition.

Figure 3-9: IgG bands on reduced SDS-PAGE gel Due to complete or incomplete reduction of disulphide bonds in IgG up to five detectable bands occur. The highest relative quantity is that of completely reduced heavy chain at ~55 kDa and light chain at ~22 kDa.

~155

~130

~75

~55

~22

250

150

100

75

50

37

25

20

IgG kDa #1 #2 Mw kDa

Lane: 1 2 3 4 5 6 7 8 9 10 11 12

Lane 10

- CHAPTER 3 -

- 34 -

3.2.9. ELISA

Enzyme-linked immunosorbent assay, or ELISA, was used to analyse the IgG subclass

concentrations in samples to compare with the developed Biacore IgG subclass distribution

assay. Peliclass human IgG subclass kit (Sanquin) was used for the measurements. The kit

contained strips of wells, pre-coated with specific monoclonal anti-human subclass antibodies.

Six strips of eight wells existed for every subclass. For each experiment three strips for each

subclass was used giving 96 wells in total. The calibration curve had five points in duplicates, two

blanks and one control in duplicate leaving space for five samples in duplicates as illustrated in

Figure 3-10. If all six strips were used in one analysis this would leave space for 17 samples.

Figure 3-10: Illustration of human IgG subclass ELISA kit set-up. a) Calibration curve. b) Blanks. c) Control sample. Five samples in duplicates can be analysed at once.

The product protocol for the kit was followed. Due to the different abundances of the

different subclasses different ranges of standards were used. Following a dilution scheme the

calibrator was diluted to eight points, ranging from 10000 times to 1280000 times dilution. The

five most diluted were used for IgG1 and the five least diluted were used for IgG2-4, see Table

3-5.

# Dilution IgG1 IgG2 IgG3 IgG4

ng/ml ng/ml ng/ml ng/ml

1 1:10000 - 368 45 59

2 1:20000 - 184 22 29

3 1:40000 - 92 11 15

4 1:80000 81 46 6 7

5 1:160000 41 23 3 4

6 1:320000 20 - - -

7 1:640000 10 - - -

8 1:1280000 5 - - -

Table 3-5: IgG subclass concentrations in ELISA calibrator Concentrations of IgG1-4 in diluted standards for IgG1-4 in Peliclass human IgG subclass kit according to manufacturer.

a a a a a a a a

a a a a a a a a

a a a a a a a a

a a a a a a a a

a a a a a a a a

b b b b b b b b

c c c c c c c c

α-hIgG1 α-hIgG2 α-hIgG3 α-hIgG4


- 35 -

Samples and control sample were diluted 240000 times for IgG1 strips and 30000 times for

IgG2-4 strips. As the kit was only intended for plasma samples and not purified IgG the purified

process samples were also prediluted 1, 2 and 4 times in order to not exceed the calibration

curve. Also the HRP-conjugated secondary antibody had individual dilutions for each IgG

subclass strip: 1:500, 1:3000, 1:2000 and 1:1000 for IgG1 to IgG4 respectively.

Wells were washed four times with wash buffer.

100 μl of calibrators, control sample and samples were added to their intended wells.

Incubated for 1 hour at 37°C.


100 μl of specifically diluted HRP-conjugated antibodies were added.

Incubated for 1 hour at 37°C.


100 μl of ABTS-substrate diluted in substrate buffer were added to all wells.

Incubated for 30 minutes at room temperature.

50 μl stop solution were added to all wells.

Plates were read in a SPECTRA Max PLUS 384 plate reader at 414 nm.

4-parameter standard curves were plotted and fitted. Individual evaluation files were created

for each IgG subclass with one standard curve each. The software calculated concentrations,

taking dilutions into consideration, giving the individual subclass concentrations for the control

sample and process samples.

- RESULTS -

- 37 -

Chapter 4

4Results

4.1. Total IgG concentration assay

4.1.1. Evaluations of reagents for total IgG concentration assay

The conditions used for the immobilization and regeneration of the antibody used was

already optimized and performed according to instructions from the manufacturer. The antibody

α-hIgG was diluted to 20 μg/ml in 10 mM sodium acetate pH 5.0 pre-concentration buffer and

injected for 6 minutes, typically resulting in an immobilization level of 10000 RU. Regeneration

was performed with 3M MgCl2 for 30 seconds according to the product protocol.

4.1.2. Assay development total IgG concentration

Initially the method to use a slope (RU/s) instead of the relative response (RU) as a measure

of the signal was evaluated. Due to bulk effects from high protein concentrations (e.g. when

detecting IgG losses in discarded samples with high albumin level) and variable NaCl levels this

approach was not suitable for the assay. In order to keep the analysis time to a minimum, the

injection time was kept to only 20 seconds. Owing to the great performance of the monoclonal

anti-human IgG antibody it was possible to use a master standard curve for at least one week of

measurements with over 1000 process samples. After samples analysis the result-file was

appended in the evaluation software with a result-file containing the standard curve, it required to

be with the same method and from the same chip, flow-cell and immobilization.

- CHAPTER 4 -

- 38 -

At least one start-up cycle was necessary to condition the surface for the analysis, especially if

a master standard curve from a prior measurement was used. One or two control samples were

evenly distributed during the analysis. For example one with a high concentration and one with a

low concentration on the standard curve.

4.1.2.1. Standard curve

The standard curve was set to start at 50 μg/ml with six 2.5-fold dilutions to approximately

0.5 μg/ml. The lower point was chosen to get a high sensitivity of the assay and the higher point

to avoid the antibodies to be saturated and thereby reducing the resolution on the standard curve

for higher concentrations, as discussed in 3.2.4. The samples were then diluted to fit on the

standard curve and at the same time eliminate pH, buffer and NaCl effects by dilution. The

standard curve for IgG can be seen in Figure 4-6.

4.1.2.2. Sample preparations

Samples were first diluted using a dilution factor based on the estimated concentration,

followed by two two-fold dilutions to increase the number of measurement points and to ensure

the sample concentrations fall within the standard curve. Samples expected to contain IgG were

diluted 200 times, samples close to the final product with estimated concentrations above 10

mg/ml were diluted 1000 times and samples expected not to contain IgG were diluted at a

minimum 10 times to detect losses and to avoid pH, buffer and NaCl effects. The effect of

dilution on samples with a high (500 mM) and low (0 mM) NaCl level is exemplified in Table 4-1

concluding that the critical samples containing IgG with dilutions above 200 times were

completely diminished from NaCl effects.

Dilutions in 150 mM NaCl (HBS-EP+)

Sample 10X 20X 40X 200X 400X 800X

500 mM NaCl 185 167 159 152 151 150

0 mM NaCl 135 142 146 150 150 150

Table 4-1: Calculated NaCl levels in diluted samples Dilutions of samples with high and low NaCl level into HBS-EP+ with 150 mM NaCl. Samples diluted 10 and 20 times have a moderately increased or decreased level from optimal which might give a positive or negative bulk, as described in section 2.3. Although for samples diluted 200 times and more all effects are diminished, these are also the sample with the most critical concentrations.

- RESULTS -

- 39 -

4.1.2.3. Assay procedure

Immobilization of 20 μg/ml α-hIgG for 6 minutes resulted in approximately 10000 RU ligand.

After conditioning start-up cycles the calibrant was injected in increasing concentrations, if a

master standard curve was not employed. Thereafter samples with increasing concentrations

within the three dilutions were injected in duplicates. Also evenly distributed control-samples

were injected. Regeneration was performed with 30 second injection of 3M MgCl2 after each

cycle. When evaluating the results one dilution giving either a too low concentration or a too high

concentration on the standard curve was excluded giving four determinations for each sample

(n=4). For samples without expected IgG, i.e. 10 times diluted samples, if possible the two most

diluted samples were chosen to avoid buffer, pH and NaCl effects. For samples containing IgG,

i.e. 200 and 1000 times diluted samples, if possible the two least diluted samples were chosen to

stay away from errors from high dilution.

To check the stability of the assay one IgG sample was injected in 1000 cycles with four

standard curves and control samples. The stability was very high with a CV of 1.34 % for the

sample. The relative responses are shown in Figure 4-1.

Figure 4-1: 1000 cycles stability check total IgG assay A test to check the stability of the assay was performed with 1000 injected sample cycles and four standard curves and control samples. After evaluation the sample had a CV of 1.34 %.

0

500

1000

1500

2000

2500

3000

3500

0 100 200 300 400 500 600 700 800 900 1100

Binding stability

Re

lati

ve r

esp

on

se -

sta

bilit

y

RU

Cycle number

Calibration

Control

Sample

Startup

0

500

1000

1500

2000

2500

3000

3500

0 100 200 300 400 500 600 700 800 900 1100

Binding stability

Re

lati

ve r

esp

on

se -

sta

bilit

y

RU

Cycle number

Calibration

Control

Sample

Startup

CV=1.34 %

- CHAPTER 4 -

- 40 -

4.1.2.4. Quick, in-process analysis

An optional assay for quick, in-process analysis was also developed. This can be utilised if a

master standard curve has not previously been created and a rapid concentration determination is

required, for example before, during and after the concentrating ultrafiltration steps. The

injection time was reduced to 5 seconds, with a flow-rate of 20 μl/min to make sure that the

lower limit of injection volume of 2 μl was overcome. Only two concentrations of calibrant were

injected, 12.5 μg/ml and 50 μg/ml and a linear standard curve was constructed, Figure 4-3.

Samples were injected with dilutions of 100, 200 and 400 times. Furthermore, the regeneration

time was also decreased to 20 seconds and the pre-dip was removed. This reduced the cycle time

from 195 to 160 seconds, which gave a concentration result in less than 14 minutes for one

sample with three dilutions and two calibration points, Figure 4-2.

Figure 4-2: Sensorgram from quick, in-process analysis The sensorgram with only two standard points and 5 seconds sample injection and 20 seconds regeneration without pre-dip resulting in a 160 seconds cycle time.

Figure 4-3: Standard from curve quick, in-process analysis The simplified linear standard curve with only two standard points for quick in-process analysis when a master standard curve is not yet available.

-100

300

700

1100

1500

0 20 40 60 80 100 120 140 160

Adjusted sensorgramRU

Resp

on

se (

0 =

baselin

e)

sTim e

0

200

400

600

800

1000

0 10 20 30 40 50

Rela

tive R

esp

on

se

RU

Concentration µg/ml

IgG

Sample injection, 5 sec 50 μg/ml 20 sec 12.5 μg/ml Regeneration

Re

lati

ve

Re

spo

nse

(R

U)

- RESULTS -

- 41 -

4.1.3. International reference material calibration for IgG standard

The value transfer described in section 3.2.4 was applied on the hIgG standard (target

material) utilised in the total IgG concentration assay in order to calibrate the assay. The standard

used for total IgG concentration assay (hIgG) was a reconstituted pure IgG from Sigma. 10 mg

of lyophilized powder was reconstituted in 5.0 ml HBS-EP+ and the concentration set to 2

mg/ml, it was frozen in aliquots of 100 μl and thawed when ready to use. The reconstitution of

international reference material gave an IgG concentration of 9.26 mg/ml (CR), see Table 4-2.

Reconstitution Reference Material

Concentration IgG, mg/ml C'R 9,1700

Vial + stopper, g 6,9164

Vial + stopper + water, g 7,9066

water, g Mwater 0,9903

Correction factor R 1,009795

Concentration IgG, mg/ml CR 9,2598

Concentration IgG, μg/ml CR 9259,82

Table 4-2: Reconstitution of reference material for total IgG value transfer C'R was the certified concentration of IgG in ERM-DA470k/IFCC and CR was the IgG concentration after reconstitution [14].

In Figure 4-4 are the standard curves for the three days of measuring; each measurement

resulted in two curves, slight variations between the curves were present for the higher

concentrations. As described in section 3.2.4, the dilution factor FT2 of the samples were plotted

against the relative concentration factors FR attained from interpolation of sample responses on

standard curve. Linear regression with intercept set to zero, Figure 4-5, gave the slopes 0.2001,

0.1995 and 0.1905 for day #1, day #2 and day #3 of measurements respectively.

Figure 4-4 (left): Standard curves for reference material IgG value transfer The standard curves for the three days of measuring showed slight variations for the higher concentration.

Figure 4-5 (right): Linear regressions for value transfer of IgG concentration Plotted results from: day #1 (), day #2 (), day #3 (▲). Linear regressions have the

equations: XY 2001.01 )9999.0( 2 R , XY 1995.02 )9935.0( 2 R

and XY 1905.03 )9947.0( 2 R . The slope is equal to the ratio of target

material concentration and reference material concentration.

0

500

1000

1500

2000

2500

3000

3500

4000

0 0,12 0,24 0,36 0,48 0,6

Re

lati

ve

Re

sp

on

se

RU

Concentration %

IgG RM

0,0000

0,0250

0,0500

0,0750

0,1000

0,1250

0,1500

0,1750

0,2000

0,0000 0,2500 0,5000 0,7500 1,0000

Re

lati

ve

co

nc

. Fa

cto

r, F

R

Dilution factor, FT2

- CHAPTER 4 -

- 42 -

From these slopes the new hIgG concentrations were calculated giving a mean value of 1.82

mg/ml, when the previous value was set to 2 mg/ml, shown in Table 4-3. The CV of the value

transfer was 2.73 % (n=6). The control sample had on average results of 99 % compared to

expected with CV of 1.48 %. The transfer factor for transformation of results from

measurements done prior to this calibration was calculated to 0.9107.

Results IgG value transfer Result conc. Concentration

Slope: Intercept: Control: in mg/ml Reference

Day #1 0,2001 0,0000 1,0112 1,8529 material 9,2598 mg/ml

Day #2 0,1995 0,0000 0,9829 1,8473

Day #3 0,1905 0,0000 0,9900 1,7640

Previous hIgG conc. = 2 mg/ml

Mean: 0,1967 0,9947 1,8214 New hIgG conc. = 1,82 mg/ml

Stand. Dev. 0,0054 0,0148 0,0498 Transfer factor

CV %: 2,7340 1,4833 2,7340 TF = 0,9107

Table 4-3: Results value transfer from reference material to hIgG Value transfer from the international reference material to hIgG resulted in a new IgG concentration of 1.82 mg/ml in the standard used in the study with a CV of 2.73 %. This also resulted in a transfer factor of 0.9107.

4.1.4. Results total IgG assay on plasma-derived process samples

The total IgG concentration assay described in section 4.1.2 with the protocol in Appendix C

was performed on samples from an IgG purification process. The samples were produced in lab-

scale from the starting plasma to the final IgG essentially according to the process outlined in

Figure 2-1. Two 96-well microplates were analysed on the same instrument, chip and

immobilization with triplicate injections of all samples. Measurements were done prior to the

value transfer from international reference material described above in section 4.1.3 and all

concentrations were multiplied with the transfer factor 0.9107 to give the real concentrations

presented here. Figure 4-6 shows the standard curve used for the measurements of IgG samples;

the average of three measurements was used. The regeneration of α-hIgG worked very well.

- RESULTS -

- 43 -

Figure 4-6: Human IgG standard curve The standard curve was the average of three measurements (), adjusted for new concentrations after value transfer from international reference material.

Two control samples were analysed, one with high and one with low concentration. The

recovery was on average 98.8 % and 100.7 % compared to expected with CV of 0.7 % and 0.9 %

respectively as seen in Table 4-4. Clearly stating the analysis and regeneration was successful.

Concentration Measured concentration

Compared to expected

μg/ml μg/ml CV % (recovery)

High control 18,2 18,0 0,74 98,8 %

Low control 1,17 1,17 0,90 100,7 %

Table 4-4: Control samples total IgG assay The high and low control sample had very good calculated concentration compared to expected, with CV‟s of 0.74 and 0.90 % respectively.

Below, in Table 4-5, the results from 26 samples from an IgG purification test are presented.

All samples were analysed in triplicates and three dilutions. One high or low dilution was

excluded leaving, if available, six determinations for each sample. The CV‟s are very good with

only two samples over 5 % and two thirds of the samples below 3 %. Also included are the

results from the combined biuret and SDS-PAGE analyses for comparison.

0

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2000

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0 10 20 30 40 50

Rela

tive R

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RU


IgG

- CHAPTER 4 -

- 44 -

Results total IgG concentration assay A

#

Purification step Fraction / Sample

Measured concentration

Biuret and SDS-PAGE B

Initial dilution (mg/ml)

CV% (n=6) (mg/ml)

1a Start Plasma pool 200 7,8 4,7 11 2a Pre-treatment 200 8,7 1,5 ---- D

3a Filter Permeate 200 7,7 1,7 11 4a FVIII S4FF FVIII fraction 10 0,01 7,6C 0,0 5a FIX, Alb, IgG fraction 200 3,4 2,4 4,0 7a Filter Permeate 200 3,5 2,2 3,6 8a FIX DEAE Alb, IgG fraction 200 2,8 1,7 4,4 9a FIX fraction 10 0,10 3,7 0,0 10a Alb UF1 Retentate 200 7,3 2,9 9,5 11a Filter Permeate 200 7,2 2,0 9,1 12a Alb Sx-G25 Alb, IgG fraction 200 3,6 2,0 4,2 13a Euglobulin precipitation Supernatant 200 3,6 2,9 2,7 14a Filter Permeate 200 3,6 1,8 3,8 15a Alb DEAE IgG fraction 200 2,3 1,8 2,6 16a Alb fraction 10 0,14 4,3 0,0 17a Discarded fraction 10 0,08 5,9C 0,5 18a IgG UF1 Retentate 200 4,7 2,2 3,8 19a Filter Permeate 200 4,5 2,6 4,2 20a IgG QFF IgG fraction 200 2,0 1,7 2,4 21a IgG UF2 Retentate 1000 34,2 3,1 31 22a Filter Permeate 1000 30,9 3,8 31 23a IgG CM IgG fraction 200 4,6 2,7 4,6 24a IgG UF3 Retentate 1000 28,8 3,3 29 25a Formulation 1000 27,0 3,7 31 26a Sterile filtration Permeate 1000 24,7 1,3 28

Table 4-5: Results total IgG concentration assay A The samples were produced in lab-scale, essentially according to the plasma fractionation process outlined in Figure 2-1. B Concentration calculated from biuret total protein concentration multiplied with SDS-PAGE IgG purity in percentage, giving low accuracy and sensitivity. C Due to low concentration or NaCl effects all but one dilution was excluded giving n=3 for these samples. Final IgG product (#27a) was at the time unavailable for analysis.

- RESULTS -

- 45 -

By comparing the values from the total IgG concentration assay with values calculated from

total protein biuret measurements multiplied with IgG purity estimated by SDS-PAGE, in Figure

4-7, the correlation was investigated. This shows a fairly good correlation with a slope of 1.04

from linear regression (R2 = 0.98). The compared values from biuret and SDS-PAGE had a high

degree of uncertainty for several reasons. First, biuret alone is a highly insensitive and non-

specific method. Second, the purity in percentage is only determined by estimation of peak area

from a scanned SDS-PAGE gel. Third, by combining these two methods further increases the

uncertainty of the approach.

Figure 4-7: Correlation between Biacore IgG results and biuret * SDS-PAGE results The concentrations from the total IgG assay developed here was compared with concentrations calculated from total protein concentration from biuret measurements multiplied with an estimated purity of IgG from SDS-PAGE analysis. The concentrations correlate well with a slope of 1.04 with R2-value of 0.98 from linear regression with intercept set to zero.

Samples from two steps in the process seemed to interfere more with the analysis than others.

Samples containing solvent and detergent chemicals added for virus inactivation generally

resulted in an increase of the baseline but they were possible to analyse. The other sample was a

discarded fraction from Alb DEAE Sepharose (sample #17a). This sample generally gave a very

high response during injection which disappeared directly after the injection was finished,

followed by a quick dissociation; suggesting it was mostly unspecific binding.

Also worth to mention is that the IgG purification test results presented here, did not reach

the intended final concentration of 50 mg/ml during the process. However, the total IgG assay

was successfully assessed on other purified samples where the intended final concentration was

obtained (data not shown).

As per the approved control samples and the good correlation with alternative methods the

results from the total IgG concentration assay was considered a successful analysis.

y = 1,0371x

R2 = 0,9785

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35 40Biacore assay IgG (mg/ml)

Biu

ret

* S

DS

-PA

GE

(mg

/ml)

.

- CHAPTER 4 -

- 46 -

4.2. IgG subclass distribution assay

4.2.1. Evaluations of reagents for IgG subclass distribution assay

4.2.1.1. Immobilization and activity test of four monoclonal antibodies

Four monoclonal anti-IgGSc antibodies from The Binding Site were evaluated to see how well

they were suited be used as antibodies in Biacore for the IgG subclass distribution assay. Prior to

immobilization, a pH scouting was performed on α-hIgG1a with 10 mM sodium acetate pH 4.0,

4.5, 5.0 and 5.5 as described in section 3.2.1. The result seen in Figure 4-8 clearly shows that pH

5.0 should be the most appropriate for immobilization as it was the buffer that gives a sufficiently

high response and still having the most neutral pH in order to give the mildest treatment to the

ligand. Hence, the immobilizations of all anti-IgGSc antibodies were done in pH 5.0 as they were

assumed to have similar properties.

50 times dilution of the antibodies (stock concentration 1.0 mg/ml in 100 mM tris-saline pH

8.2 with 0.099 % sodium azide) gave an antibody concentration of 20 μg/ml with only 2 mM tris-

saline and 0.002 % sodium azide, known to be low enough to not interfere with the

immobilization. Immobilization using 7 minutes injection time resulted in an immobilization level

of 9000 – 10000 RU for the four anti-IgGSc antibodies, sensorgrams can be seen in Figure 4-9.

Figure 4-8 (left): pH scouting α-hIgG1a A pre-concentration buffer with pH 5.0 seemed to be the most appropriate from the pH scouting.

Figure 4-9 (right): Immobilization anti-IgGSc antibodies Immobilization for 7 minutes resulted in levels of 9000 – 10000 RU for the four antibodies.

-5000

0

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10000

15000

20000

-50 0 50 100 150 200 250 300 350 400


Re

sp

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se

lin

e)

sTime (0 = baseline)

10 mM Acetate 4

10 mM Acetate 4,5

10 mM Acetate 5

10 mM Acetate 5,5

30000

35000

40000

45000

50000

55000

60000

65000

70000

0 200 400 600 800 1200 1600 2000

SensorgramRU

Resp

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sTime

Fc=1

Fc=2

Fc=3

Fc=4

pH 4.0 pH 4.5

pH 5.0

pH 5.5

α-hIgG1-4 EA

EDC/NHS

Re

spo

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(R

U)

Re

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Re

spo

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(RU

)

- RESULTS -

- 47 -

An initial test with a 60 seconds injection of hIgG, containing mostly IgG1 and IgG2 and less

IgG3 and IgG4, indicated an inactive or lesser active anti IgG1 antibody. The test gave a

responses of 7, 1018, 60 and 168 RU respectively on α-hIgG1-4 with IgG1 expected to give the

highest due to the higher abundance. Further, a new immobilization of α-hIgG1a with a 60

seconds injection of hIgG1 only gave 1.8 RU indicating an inactive antibody under these

circumstances.

An inverted set-up with hIgG1 as the ligand and α-hIgG1a as the analyte was performed.

After pH scouting of hIgG1 a 10 mM sodium acetate pH 5.5 was chosen and immobilization

gave 19542 RU. In the set-up, injection of α-hIgG2 was included to check cross-reactivity and α-

hIgG was included as a positive control to ensure hIgG1 activity after immobilization. α-hIgG1a

and α-hIgG2 showed no binding with 0.6 and 0.4 RU respectively and the control α-hIgG was

positive with 1601 RU response, concluding that hIgG1 was active as a ligand but α-hIgG1a does

not interact to give a visible response.

These initial tests indicated that α-hIgG1a was not sufficiently active to be used in the assay.

The antibodies for the remaining three IgG subclasses seemed to have appropriate activity and

affinity for the assay. All four reagents were further investigated in section 4.2.1.2 below.

4.2.1.2. Cross-reactivity of anti-IgGSc antibodies with capture antibodies

In order to check their activity and the cross-reactivity for different IgG subclasses the capture

set-up as described in section 3.2.1 was performed in two ways, both by capturing the anti-IgGSc

antibodies and by capturing the hIgGSc itself. Additionally the results were also supported by

immobilization of each anti-IgGSc antibody with hIgG subclasses as analytes.

The antibodies immobilized were α-hIgG (9640 RU) and anti-mouse IgG (12352 RU) to

capture hIgGSc and α-hIgGSc (from mouse) respectively. Regeneration of α-mIgG was done

with glycine pH 1.7. An example of a sensorgram from the capture set-up is displayed in Figure

4-10 where hIgG3 was captured by α-hIgG and only α-hIgG3 showed a positive result. The

results from these three cross-reactivity experiments are displayed in Table 4-6, Table 4-7 and

Table 4-8. A green mark indicates clearly positive interaction and a yellow a possibly positive

interaction.

- CHAPTER 4 -

- 48 -

Figure 4-10: Cross reactivity hIgG3 The left graph shows first the response of about 4000 RU for the capture of hIgG3. Thereafter the positive response for α-hIgG3 is seen (blue). The right enlargement with a new baseline shows no binding for α-hIgG1, α-hIgG2 and α-hIgG4.

Capture Analyte (RU)

Antibody RU hIgG1 hIgG2 hIgG3 hIgG4

α-hIgG1a 1152,0 50,5 37,2 45,1 35,6

α-hIgG2 1058,0 40,8 46,2 41,8 32,2

α-hIgG3 738,2 40,8 30,7 260,7 33,0

α-hIgG4 857,9 37,2 29,6 36,6 359,3

Table 4-6: Cross-reactivity test with capture α-hIgG1-4 and analyte hIgG1-4 A clear positive response was seen for hIgG3 when α-hIgG3 was captured and for hIgG4 when α-hIgG4 was captured.


Antibody RU α-hIgG1a α-hIgG2 α-hIgG3 α-hIgG4

hIgG1 3788,7 31,7 16,2 8,9 14,9

hIgG2 2997,7 11,6 110,6 -3,0 -1,7

hIgG3 3910,9 2,9 -14,7 2189,0 -3,0

hIgG4 3558,1 15,6 4,7 5,3 804,0

buffer ---- 23,0 2,4 1,0 3,2

Table 4-7: Cross-reactivity test with capture hIgG1-4 and analyte α-hIgG1-4 When hIgG1-4 was captured by α-hIgG a weak positive result was seen for α-hIgG1a and strong positive results for α-hIgG2-4. No signs of cross-reactivity.

Ligand Analyte (RU)


α-hIgG1a 8566,8 6,3 6,4 9,3 7,8

α-hIgG2 9962,9 0,1 154,6 4,5 2,5

α-hIgG3 8845,8 6,6 3,8 3819,4 9,4

α-hIgG4 8799,6 8,0 8,6 9,2 1078,2

Table 4-8: Cross-reactivity test with ligand α-hIgG1-4 and analyte hIgG1-4 As α-hIgG1-4 were immobilized to 8500-10000 RU positive interaction was only seen for hIgG2-4 and no cross-reactivity was seen.

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- RESULTS -

- 49 -

All together the experiments gave the same four conclusions: α-hIgG1a does not have enough

activity for a Biacore assay, α-hIgG2 has a low activity but it was evidently detectable using

Biacore; α-hIgG3-4 have a high activity and finally that none of the antibodies show any cross-

reactivity to other IgGSc than they were intended for.

This concluded that α-hIgG1a was not a suitable antibody for the assay and will hereby be

excluded.

4.2.1.3. Evaluation of polyclonal sheep anti-human IgG1 antibody

To find a suitable replacement for the inadequate monoclonal antibody α-hIgG1a for binding

of hIgG1 in the subclass distribution assay a polyclonal anti-hIgG1 antibody was evaluated. The

polyclonal sheep anti-human IgG1 antibody from The Binding site denoted α-hIgG1poly was

immobilized on all flow-cells with pH 5.0, determined from pH scouting, to approximately 8000

RU. Each hIgGSc were injected for 60 seconds in two concentrations (12.5 and 25 μg/ml) with

regeneration by 30 second glycine pH 2.0. The antibody gave a high response to the intended

hIgG1 but also expressed a rather high cross-reactivity to hIgG4 and a moderate cross-reactivity

to hIgG3 as seen in Figure 4-11 and Table 4-9. Hence, the polyclonal antibody was excluded as a

potential antibody for the assay.

Figure 4-11: Sensorgram cross-reactivity test α-hIgG1poly Each hIgGSc was injected in two concentrations, 25.0 and 12.5 μg/ml. The highest response was as expected for hIgG1 but also unexpectedly hIgG3-4 showed a fairly high response.

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- CHAPTER 4 -

- 50 -

Analyte Conc. (μg/ml)

Response (RU)

cross-reactivity

Comment

hIgG1 25,0 1400 100,0% Set to 100 %

12,5 1100 100,0% Set to 100 %

hIgG2 25,0 16 1,1% No cross reactivity

12,5 10 0,9%

hIgG3 25,0 135 9,6% Moderate cross-reactivity

12,5 95 8,6%

hIgG4 25,0 260 18,6% High cross-reactivity

12,5 160 14,5%

Table 4-9: Results cross-reactivity test α-hIgG1poly The results from Figure 4-11 are summarized and cross-reactivity calculated. The responses from hIgG1 were set to 100% and other IgGSc compared to these. The highest cross-reactivity was seen for hIgG4 with over 15 %. Therefore this polyclonal α-hIgG1 antibody was excluded from the study.

4.2.1.4. Evaluation of two monoclonal anti-human IgG1 antibodies

To complete the reagents for the IgGSc distribution assay two monoclonal anti-human IgG1

antibodies from Invitrogen were evaluated, denoted α-hIgG1b (clone HP6069) and α-hIgG1

(clone HP6070). Both antibodies, immobilized to 11000 and 9000 RU with 20 μg/ml in sodium

acetate pH 5.0, provided stable interaction and no cross-reactivity to hIgG2-4 as summarized in

Table 4-10 with sensorgrams in Figure 4-12. The regeneration was successful with 12.5 mM

NaOH.

Figure 4-12: Sensorgram α-hIgG1 (clone HP6070) cross-reactivity test Positive interaction for hIgG1 on α-hIgG1 immobilized to 8000 RU. All other hIgGSc showed no interaction. 12.5 mM NaOH regeneration was successful.

Table 4-10: Results α-hIgG1 (clone HP6070) cross-reactivity test Positive binding without any cross-reactivity was seen for the two monoclonal anti-hIgG1 antibodies.

As both displayed similar activity and no cross-reactivity α-hIgG1 (clone HP6070) was chosen

primarily on the slightly higher responses compared to α-hIgG1b (clone HP6069), despite the

lower immobilization level.

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hIgG2 25ug/ml

hIgG3 25ug/ml

hIgG4 25ug/ml

Ligand Analyte


α-hIgG1b (HP6069) 11066 121 11 8,2 14

α-hIgG1 (HP6070) 8850 135 11 7,6 13

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hIgG1 25ug/ml

hIgG2 25ug/ml

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- RESULTS -

- 51 -

4.2.1.5. Complete regeneration scouting for anti-human IgG2

Monoclonal antibody α-hIgG2 was scouted for regeneration conditions using the protocol

described in section 3.2.3.1. Five surfaces on two CM5 sensor chips were immobilized with α-

hIgG2 to 9000-10000 RU. All the regeneration solutions were tested with increased condition

strength.

All the overlay-plots are included in Appendix A. The overlay-plot for NaOH, in Figure 4-13

below, shows the solution giving the most promising regeneration. It shows that as the

concentration reaches 10 mM NaOH the surface was being regenerated as the accumulated

analyte was removed and the binding response was regained. For NaOH concentrations of 25

mM and higher, the binding response was lost even though the baseline drops, the concentration

was therefore too high.

In order to verify these findings and to optimize the regeneration further, over 30 cycles with

the same condition was performed and displayed in Figure 4-14. 10 mM NaOH regeneration for

30 seconds was shown not to regenerate completely as the baseline was increasing for every cycle

due to accumulating analyte. With 15 mM NaOH solution the binding response was decreasing

quite much compared to the initial response.

Finally, a slightly weaker concentration of 12.5 mM but with a longer injection time showed a

reasonably good regeneration and a response recovery of approximately 65 %. The working

regeneration condition was also verified on all chosen anti-human IgGSc antibodies with

response recoveries between 40 % (IgG4) and 83 % (IgG3), data not shown.

Figure 4-13: Regeneration scouting of α-hIgG2 with increasing concentration NaOH For the lower concentrations of NaOH (1 and 5 mM) the sample response was decreasing while the baseline was increasing, suggesting incomplete regeneration with analyte accumulation. As the concentration reached 10 mM NaOH the sample response increased to a probable stable level while the baseline correspondingly decreased. For higher concentrations (above 25 mM) both the sample response and baseline were decreasing, suggesting too harsh regeneration conditions. A regeneration condition of 10 mM NaOH was therefore investigated further.

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1mM 5 mM 10 mM NaOH NaOH NaOH

25mM 50 mM 75 mM NaOH NaOH NaOH

- CHAPTER 4 -

- 52 -

Figure 4-14: Regeneration performance of α-hIgG2 with different NaOH conditions Over 30 cycles of various regeneration conditions were assessed. 10 mM NaOH for 30 seconds was not enough as the analyte seems to accumulate on the surface (see B). An increase to 15 mM NaOH had a larger decrease in sample response. Finally 12.5 mM NaOH with a 60 second injection appeared to be the best regeneration condition for α-hIgG2 with a response recovery of 65 %.

4.2.2. Assay development IgG subclass distribution

The approach for this assay was to use all four flow-cells serially with one antibody

immobilized in each flow-cell specific to one of the four IgG subclasses. The sample and

calibrator will be injected and regenerated in all flow-cells simultaneously and only one calibrator

containing normal IgG consisting of a normal distribution of all subclasses shall be used. This

was to minimize the analysis time and maintain the subclass distribution and eliminate faults due

to different dilutions.

Two difficulties were evident with this approach. First, to have four different antibodies that

regenerate under the same conditions. Second, due to the normal distribution of the subclasses in

total IgG the four different calibration curves will be dissimilar with much higher concentrations

of IgG1 and IgG2 compared to IgG3 and IgG4.

As seen in the evaluation of antibodies for the assay in section 4.2.1, the chosen antibodies

have very different affinity and activity. The antibodies against IgG1 and IgG2 have a lower

activity than the antibodies against IgG3 and IgG4 which therefore desirably might lead to a

similar response in the four flow-cells. Shown in section 4.2.1.5, the antibodies chosen all

regenerate well with a 60 seconds injection of 12.5 mM NaOH solution.

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A) B)

- RESULTS -

- 53 -

4.2.2.1. Standard curve

The IgG solution used as the calibrator was from the ELISA-kit (here denoted IgGSc-

standard) contains 3.62 (54.1 %), 2.34 (35.0 %), 0.53 (7.9 %) and 0.20 (3.0 %) mg/ml of IgG1-4

respectively according to the value transfer from international reference material performed in

section 4.2.3. When using six dilutions of the calibrator ranging from 40 times dilution to 1280

times dilution with a two-fold dilution between; the concentrations of the four calibration curves,

one for each antibody and flow-cell, were as shown in Table 4-11. As expected they were highly

dissimilar with approximately 3 to 90 μg/ml for IgG1 and 0.2 to 5 μg/ml for IgG4.

hIgG1 hIgG2 hIgG3 hIgG4

dilution μg/ml μg/ml μg/ml μg/ml

1280 2,8 1,8 0,4 0,2

640 5,7 3,7 0,8 0,3

320 11,3 7,3 1,7 0,6

160 22,6 14,6 3,3 1,2

80 45,2 29,2 6,6 2,5

40 90,4 58,5 13,3 5,0

Table 4-11: Calibration curves IgG subclasses Concentrations for the four IgG subclasses in IgGSc-standard after dilutions. The concentrations are shown after the value transfer from international reference material in section 4.2.3.

4.2.2.2. Sample preparation

The samples analysed with the assay were varying from normal human plasma to final

purified, concentrated and formulated IgG product. This puts a high demand on the range of the

assay from high to low concentrations of the subclasses. It was previously known that in final

products of intravenous IgG the distribution of IgG3 and IgG4 will be even lower than in

normal plasma, as low as 1.0 % IgG3 and 0.5 % IgG4 was specified by several other

manufacturers of IVIG (Octapharma, Sweden; Sanquin, The Netherlands and CSL, Australia).

Due to the lower concentration of IgG3 and IgG4 in the final product as well as a higher

concentration of total IgG in these samples the dilutions have to be altered between samples

from different stages in the process.

Suitable preparations were a first dilution of the samples, followed by four two-fold dilutions

thereafter to get a large concentration range for all subclasses. For samples not expected to

contain any IgG in the process, i.e. to detect losses, lowest possible dilution was 10 times to avoid

pH, NaCl and buffer effects. For samples late in the process with a total IgG concentration

below 5 mg/ml a first dilution of 20 times was appropriate. For final IgG preparations with

concentrations around 40-50 mg/ml, a dilution of about 160 times was needed (not applicable on

the samples analysed here). Finally, for all other samples, including start plasma, a first dilution of

80 times resulted in reasonable concentrations suitable for the calibration curves.

- CHAPTER 4 -

- 54 -


Immobilization was done through the immobilization wizard with 20 μg/ml for all antibodies

and the contact time 11, 10, 7 and 7 minutes for α-hIgG1-4 respectively. This resulted in

immobilization levels of 7970, 11153, 9536 and 8791 RU for α-hIgG1-4 respectively. The assay

was optimized for a 120 seconds injection to save time and a 5 μl/min flow-rate to save reagents.

The regeneration was divided into two separate injections due to IgG1 and IgG2 demanding

stronger regeneration than IgG3 and IgG4. The first injection was 60 seconds of 12.5 mM

NaOH with 10 μl/min onto flow-cell one and two, and the second injection was 30 seconds of

12.5 mM NaOH with 10 μl/min onto all four flow-cells. These conditions were found to be the

most appropriate for these antibodies and samples. As mentioned in section 4.2.1.5, α-hIgG4 has

a great loss of activity during the first few cycles and therefore demand at least 10 start-up cycles

to stabilize.

The six dilutions of calibrant were injected with increasing concentrations onto all flow-cells;

at least once first and once last and preferably also distributed evenly if many samples were

analysed, see sensorgrams in

Figure 4-15. One conditioning cycle (dummy cycle) with only running buffer as sample but

with normal regeneration was required after the last cycle of each sample, the one with the

highest concentration, in order to completely regenerate the surface before the next sample starts.

A control sample, preferably the same used as start-up sample should be included quite

frequently to ensure the accuracy of the assay. Here, a sample similar to the standard with known

concentrations of IgGSc, called IgGSc-control, was used with a 320 times dilution giving

concentrations of 11.42, 7.33, 1.62 and 0.63 μg/ml for IgG1-4 respectively.

Figure 4-15: Sensorgrams standard curves IgG1-4 All sensorgrams have the same scale showing injection of six dilutions of standard and regeneration. For IgG1-2 there were two regeneration injections and for IgG3-4 only the latter.

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- RESULTS -

- 55 -

4.2.2.4. Evaluation

In order to evaluate the results it was required to use the so called trend that is incorporated in

Biacore T200 evaluation software. This takes into consideration the decrease of response

between the first calibration curve and the final calibration curve that here occurs due to non-

optimal regeneration of the immobilized antibodies.

The four IgG subclasses needs to be evaluated separately as they each have their individual

calibration curve in each flow-cell and the software does not support multiple calibration curves

to be used. Four different evaluation files were used where the concentrations of the calibrator

and control were changed for each one to accommodate for the concentration of that specific

IgG subclass and flow-cell. During the evaluation, two dilutions giving either a too high or a too

low concentration on the standard curve were excluded leaving a minimum of three

determinations (n=3) if possible. Alternatively, by using relative concentration in percentage

simplifies the evaluation by only requiring one evaluation file with four standard curves; further

described in section 3.2.6 and exemplified in the protocol in Appendix D.

4.2.3. International reference material calibration IgGSc-standard

The value transfer described in section 3.2.4 was applied on the hIgGSc-standard (target

material) utilised in the IgG subclass distribution assay in order to calibrate the assay. The

standard used for IgG subclass distribution assay had the following specified concentrations for

each IgGSc: 6.51, 3.68, 0.449 and 0.586 mg/ml according to the manufacturer. The individual

concentrations of IgGSc were not specified in the reference material but an assignment had been

performed by Williams et al for the American Society of Clinical Chemists in 2009 [34]. This

assignment was done with a minimum of 54 measurements for each IgG subclass from the

preceding international reference material CRM470 [35]. The concentrations before and after

reconstitution of reference material are presented in Table 4-12.

Reconstitution Reference Material IgG1 IgG2 IgG3 IgG4

Concentration IgGSc, mg/ml C'R 4,771 3,488 0,523 0,373




Correction factor R 1,009795 IgG1 IgG2 IgG3 IgG4

Concentration IgG, mg/ml CR 4,8177 3,5221 0,5281 0,3766

Table 4-12: Reconstitution of reference material for IgGSc value transfer C'R was the certified concentrations of IgG subclasses in ERM-DA470k/IFCC according to the assignment by Williams et al. and CR was the IgGSc concentrations

after reconstitution [34].

As described in the assay development in section 4.2.2 the assay requires a trend calibration

due to the decrease in response for each cycle of regeneration. This is clearly seen in the standard

curves for reference material in Figure 4-16 for the four subclasses. Each measurement resulted

in two curves, one before and one after the samples and Figure 4-16 shows all three days of

measuring. The decay in response was obvious and the trend calibration tool was therefore used.

- CHAPTER 4 -

- 56 -

Figure 4-16: Standard curves for reference material IgGSc value transfer The standard curves for the four IgG subclasses. The first and second curves from the top are from day 1, third and fourth from day 2 and the last two from day 3 of analysis. For each day the first curve is before and the last curve is after sample analysis. Trend calibration tool was used to take into account the decrease between the curves.

As described in section 3.2.4, the dilution factor FT2 of the samples were plotted against the

relative concentration factors FR attained from interpolation of sample responses on the standard

curves. The linear regressions with intercepts set to zero are displayed in Figure 4-17, which gave

the mean slopes 0.7508, 0.6642, 1.0044 and 0.5260 for IgG1-4 respectively.

Figure 4-17: Linear regressions for value transfer of IgGSc concentration Plotted results from: day #1 (), day #2 () and day #3 (▲); blue = IgG1, purple = IgG2, red = IgG3 and green = IgG4. The mean slopes (β) for the three days were 0.7508, 0.6642, 1.0044 and 0.5260 for IgG1-4 respectively. The slope is equal to the ratio of target material concentration and reference material concentration, from Equation 3-5. All linear regression had an average R2 of 0.9973.

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- RESULTS -

- 57 -

From these slopes the new hIgGSc concentrations in the standard were calculated giving

mean concentrations of 3.62, 2.34, 0.53 and 0.20 mg/ml for IgG1-4 respectively, when the

previous values were set to 6.51, 3.68, 0.449 and 0.586 mg/ml, shown in Table 4-3. All CV‟s of

the value transfer were below 6.5 %. Transfer factors for transformations of results from

measurements done prior to this calibration were calculated to 0.5556, 0.6357, 1.1814 and 0.3381

for IgG1-4 respectively.

Results IgGSc value transfer

IgG1 IgG2 IgG3 IgG4 Total

Reconstituted RM, mg/ml 4,82 3,52 0,53 0,38 9,24

Distribution, % 52,1% 38,1% 5,7% 4,1%

Target Material (IgGSc-standard)

Day #1 3,62 2,34 0,53 0,20 6,69

Day #2 3,85 2,45 0,55 0,20 7,05

Day #3 3,38 2,23 0,51 0,19 6,31

New IgGSc conc., mean, mg/ml 3,62 2,34 0,53 0,20 6,68

Distribution, % 54,1% 35,0% 7,9% 3,0%

Standard deviation 0,24 0,11 0,02 0,005

CV % 6,5 4,6 3,7 2,4

Previous IgGSc-standard

concentration, mg/ml 6,51 3,68 0,449 0,586 11,23

Distribution, % 58,0% 32,8% 4,0% 5,2%

Transfer factor (TF) 0,5556 0,6357 1,1814 0,3381

Table 4-13: Results value transfer from reference material to IgGSc-standard Value transfer from the international reference material to IgGSc-standard resulted in new IgG subclass concentrations of 3.62, 2.34, 0.53 and 0.20 mg/ml for IgG1-4 respectively with CV below 6.5 %. The transfer factors were calculated to 0.5556, 0.6357, 1.1814 and 0.3381 for IgG1-4 respectively.

4.2.4. Results IgG subclass distribution assay on plasma-derived samples

The assay described above in section 4.2.2 and in the protocol in Appendix D was applied on

17 samples throughout the lab-scale plasma fractionation process as illustrated in Figure 2-1, with

results presented below. The samples were distributed from the start plasma to the final IgG

product aiming to evaluate every major step of the process to detect changes in distribution and

possible losses of any of the IgG subclasses. The four graphs in Figure 4-18 show the results

from the initial (black), middle (red) and final (blue) calibration curve; between which the trend

calibration tool interpolated curves. As seen in Figure 4-18 all except IgG3 have a fairly similar

response despite the different concentrations in total IgG.

- CHAPTER 4 -

- 58 -

Figure 4-18: Calibration curves IgG1-4 Calibration curves were analysed in the beginning (black), in the middle of the experiment (red) and after all samples (blue). The trend calibration tool takes into consideration the decrease during the experiment. The antibody α-hIgG3 showed very good activity and regeneration.

Both Figure 4-18 and Figure 4-20 clearly show the superior activity and stability of α-hIgG3

and the reduction of activity in all others during the 140 cycles in the experiment. The need of

start-up cycles is also apparent in Figure 4-20, especially for α-hIgG4 (purple) which drops from

350 RU to below 150 RU during the first 10 cycles. The baselines seen in Figure 4-19

demonstrate good regeneration for α-hIgG3 and α-hIgG4 as the baseline was only decreasing

slightly for each cycle. In contrast, α-hIgG1 and mainly α-hIgG2 sometimes has an incomplete

regeneration, with a build-up on the baseline for higher concentration of IgG1 and IgG2. Figure

4-20 demonstrate good regeneration for IgG3 and acceptable for the other subclasses.

For the last three samples that have a very high concentration of IgG1 and IgG2 but very low

concentration of IgG3 and IgG4 this effect was especially apparent (cycle 120 until the end in

Figure 4-19). Due to the aim of the assay to always inject all samples onto all flow-cells the

dilution of 80 times was essential to detect the low concentration of IgG4 whilst this leads to

extremely high concentrations of IgG1 and IgG2 complicating the regeneration.

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hIgG1 hIgG2

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- RESULTS -

- 59 -

Figure 4-19 (left): Baseline α-hIgG1-4 The absolute response of the baseline for the four flow-cells during the experiment. Results show acceptable baseline changes for all with some accumulation on surface for α-IgG1-2 for samples late in the process with very high IgG1 and IgG2 concentrations.

Figure 4-20 (right): Start-up and control samples α-hIgG1-4 The responses from start-up cycles and control samples show the need of start-up cycles due to the major decrease in response during the first 10 cycles. Results show good repeatability of responses for IgG3 and fairly acceptable responses for the others, Also, the need of trend calibration is apparent as the response for the control sample decreases during the experiment.

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α-hIgG2

α-hIgG3

α-hIgG4

α-hIgG1

α-hIgG3

α-hIgG2

α-hIgG1

α-hIgG4

Baseline Start-up and control samples

- CHAPTER 4 -

- 60 -

The control sample was represented by a 320 times dilution of the sample denoted IgGSc-

control (control sample from ELISA-kit). The specified subclass concentration values on IgGSc-

control were not calibrated against the international reference material in the same manner as

IgGSc-standard. Hence, the values from the manufacturer multiplied with the transfer factor

from the value transfer are not necessarily the correct control concentrations. Consequently, the

results from the control samples presented in Table 4-14 should be interpreted thereafter. All

concentrations seem to be approximately 20 % overestimated, but since the distribution was

almost exactly the same and the CV‟s are all below 5 % it was considered an approved control

nonetheless.

IgGSc-control Calculated CV % Compared

Conc. Distr. Conc. Distr. to expected

IgG1 11,42 54,4% 14,56 54,6% 2,85 127,4%

IgG2 7,33 34,9% 9,27 34,8% 4,49 126,5%

IgG3 1,62 7,7% 2,08 7,8% 0,36 128,3%

IgG4 0,63 3,0% 0,77 2,9% 4,52 123,0%

Table 4-14: Control samples IgG subclass distribution assay IgGSc-control was not calibrated against the international reference material. The concentrations were derived from manufacturer‟s specifications multiplied with transfer factor for IgGSc-standard. The measured distribution was almost exactly the same and the CV‟s are all below 5 % so it was considered an approved control.

The evaluated results presented in Table 4-15 give concentration in mg/ml for each IgGSc

and a sum of all subclasses together; from these concentrations the percentage distribution was

calculated. The CV‟s for all samples except three were below 4.0 % (n=3) and with an average

CV of 2.13 % for the whole experiment. Concentrations are the mean result from three dilutions.

The results showed a highly reasonable distribution in the start plasma sample with 53.4 %,

35.0 %, 7.35 % and 4.18 % IgG1 to IgG4 respectively. Throughout the process the proportion of

IgG4 in the samples were decreasing slightly and in the Q-Sepharose Fast Flow (QFF)

chromatography step, which is a strong anion exchanger, both IgG3 and IgG4 decreased. This

result was also strengthened with the discarded fraction from QFF which contained relatively

high concentrations of IgG3 and IgG4 (data not shown). Thereafter, the distribution in the final

product stayed fairly stable with only a slight decrease in IgG3 and IgG4 level.

- RESULTS -

- 61 -

Results IgG subclass distribution assay A

#

Purification step Fraction / Sample

hIgG1 mg/ml

hIgG2 mg/ml

hIgG3 mg/ml

hIgG4 mg/ml

Sum of IgG1 to 4 mg/ml

Total IgG Biacore mg/ml Subclass distribution (%)

1a Start Plasma pool 4,5 2,9 0,6 0,35 8,4 7,7

53,4% 35,0% 7,4% 4,2% 4a FVII S4FF FVIII fraction 0,00 0,00 0,00 0,00 0,00 0,01

0,0% 0,0% 0,00% 0,00% 5a FIX, Alb, IgG fraction 1,9 1,2 0,25 0,13 3,5 3,6

53,7% 35,3% 7,1% 3,8% 8a FIX DEAE Alb, IgG fraction 1,6 1,0 0,20 0,11 2,9 2,8

54,0% 35,2% 7,0% 3,8% 9a FIX fraction 0,03 0,00 0,00 0,00 0,03 0,10

88,9% 0,0% 0,0% 11,1% 10a Alb UF1 Retentate 3,9 2,6 0,50 0,28 7,2 7,3

53,7% 35,4% 6,9% 3,9% 12a Alb Sx-G25 Alb, IgG fraction 1,5 1,2 0,19 0,10 3,0 3,6

49,3% 41,1% 6,2% 3,4% 13a Euglobulin Supernatant 1,9 1,3 0,24 0,10 3,5 3,6

precipitation 53,6% 36,7% 6,8% 2,9% 15a Alb DEAE IgG fraction 1,2 0,82 0,15 0,05 2,2 2,3

53,4% 37,5% 6,8% 2,3% 16a Alb fraction 0,00 0,03 0,00 0,02 0,05 0,14

0,0% 60,0% 0,0% 40,0% 17a Discarded fraction 0,00 0,00 0,00 0,00 0,00 0,08

0,0% 0,0% 0,0% 0,0% 18a IgG UF1 Retentate 2,4 1,7 0,31 0,10 4,6 4,7

52,7% 38,3% 6,8% 2,3% 19a Filtration Permeate 2,2 1,6 0,28 0,09 4,1 4,5

52,5% 38,5% 6,7% 2,3% 20a IgG QFF IgG fraction 1,1 0,78 0,03 0,01 1,9 2,0

57,3% 40,9% 1,5% 0,29% 22a Filtration Permeate 18,7 11,7 0,45 0,06 30,9 30,9

(after IgG UF2) 60,6% 37,8% 1,5% 0,2% 24a IgG UF3 Retentate 16,9 9,3 0,34 0,05 26,5 28,8

(after IgG CM) 63,7% 34,9% 1,3% 0,17% 27a Final formulation IgG final product 10,7 6,1 0,20 0,03 17,1 24,7 B 62,9% 35,7% 1,2% 0,18%

Table 4-15: Results IgG subclass distribution assay A The samples were produced in lab-scale essentially according to the plasma fractionation process shown in Figure 2-1. B Total IgG concentration from #26a instead of #27a. It was seen that the majority of IgG3 and IgG4 were lost in the Q Sepharose FF step (between #19a and #20a) and thus retained in the discarded fraction (data not shown).

- CHAPTER 4 -

- 62 -

Also included in Table 4-15 is the results from the total IgG assay on the same samples in

section 4.1.4. By comparing these results with the summarized concentration of IgG1 to IgG4

the graph in Figure 4-21 was composed. This show a very good linear correlation between the

concentrations from the two assays.

When excluding the three highest concentrations the linear relation becomes 0.99 with a R2

value of 0.99, with intercept set to zero. When analysing the final product (i.e. the excluded

samples with high concentrations) the high IgG1 and IgG2 concentrations might vary slightly

from real concentrations due to the inadequate regeneration as discussed previously in this

section.

Figure 4-21: Correlation between summarized IgG1-4 concentration with total IgG. The thin blue line in the both graphs represents a one to one correlation. In the left graph an outlier (sample #27a) affects the correlation negatively; this point can be excluded as the corresponding total IgG value was from the prior sample #26a instead of #27a. In the right graph the three highest concentrations are excluded and the area between 0 and 10 mg/ml enlarged showing a very good linear relation with a slope of 0.99 with a R2 value of 0.99.

4.2.4.1. Results IgG subclass ELISA kit

As a comparison, ELISA measurements on IgG subclass distribution were also performed on

the final IgG preparation (#27a) as described in section 3.2.9. Due to the expected concentration

of total IgG of approximately 20 - 25 mg/ml (from measurements with Biacore and biuret in

Table 4-5) the sample was diluted 1, 2 and 4 times prior to the protocol dilutions to fall within

the standard curve (intended for normal plasma samples). The standard curves are presented in

Figure 4-22 with average optical density on the y-axis and concentration in ng/ml on the x-axis.

y = 1,0872x

R2 = 0,972

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- RESULTS -

- 63 -

Figure 4-22: Standard curves IgG subclass ELISA The standard curves for the four IgG subclasses in ELISA measurements. Evaluated in SoftMax Pro v5.4 with one evaluation file for each subclass due to different standard concentrations.

The control sample (IgGSc-control) gave concentrations within the limits of the ELISA kit.

Several of the sample concentrations for IgG3 and IgG4 were below the standard curve and thus

did not give an acceptable value. For the final IgG preparation (#26a) the sample diluted one or

two times gave a reproducible concentration and distribution, as shown in Table 4-16. Even

though the distribution correlates fairly well with that from the Biacore IgG subclass distribution

assay, the concentrations for IgG1 and IgG2 were much lower than expected. The low levels of

IgG3 and IgG4 were also evident with this method.

Sample Dilution IgG1

(mg/ml) % IgG2

(mg/ml) % IgG3

(mg/ml) % IgG4

(mg/ml) % Total

(mg/ml)

#27a 1X 7,4 59 4,8 39 0,11 0,9 0,12 1,0 12,50

#27a 2X 7,4 54 5,9 44 0,12 0,9 0,16 1,2 13,58

Control sample 6,1 55 3,9 36 0,42 3,7 0,58 5,3 11,09

(control values) 6,6 58 3,7 33 0,44 3,9 0,59 5,2 11,30

Table 4-16: Results IgG subclass ELISA, final IgG product The results for final IgG product (#27a) to compare with Biacore results in Table 4-15.

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- CHAPTER 4 -

- 64 -

4.3. Albumin concentration assay

4.3.1. Evaluations of reagents for albumin concentration assay

4.3.1.1. Polyclonal rabbit anti-human serum albumin

An in-house polyclonal anti-HSA denoted α-HSApoly was initially evaluated for immobilization, regeneration and activity. A pH scouting in

Figure 4-23, as in section 3.2.1, revealed optimal use of either pH 5.0 or 5.5 for

immobilization, pH 5.0 was chosen for standardization purposes. The antibody was diluted in 10

mM sodium acetate pH 5.0 to approximately 20 μg/ml and immobilized to a level of 11000 RU.

Injection for 60 seconds of 50 μg/ml essentially fatty-acid free human serum albumin, denoted

HSAa, gave approximately 1200 RU binding response. The regeneration was successfully

accomplished with a 30 second injection of glycine pH 2.0.

Figure 4-23: pH scouting α-HSApoly The pH scouting of α-HSApoly showed the optimal use of a pre-concentration buffer with pH 5.0-5.5. The chosen buffer was pH 5.0 for standardization purposes.

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pH 4.0

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- RESULTS -

- 65 -

4.3.1.2. Monoclonal anti-human serum albumin

A monoclonal anti-HSA from Abcam to be used in the albumin assay, instead of the

polyclonal antibody α-HSApoly unavailable to purchase, was tested for cross-reactivity and

regeneration.

Dilution to 15 μg/ml with 10 mM sodium acetate pH 5.0 and injection for 7 minutes with a

flow-rate of 5 μl/min (to save reagents) gave an immobilization level of 10000 RU. Glycine pH

2.0 in 30 seconds was chosen as regeneration condition, same as for α-HSApoly.

Cross-reactivity was assessed by injecting 100 μg/ml of hIgG, BSA and HSAa for 20 seconds.

The sensorgram in Figure 4-24 shows the response of 2200 RU for HSAa compared with the

weak response of 8 RU for BSA (in magnification). This implies a very low cross-reactivity of

BSA, although the interaction quickly dissociates and thus has a low affinity. As the process

samples do not include any BSA this shall not have any impact. More important was that the

cross-reactivity with hIgG was considered negligible and therefore possibly not interfering with

the concentration assay. These results are summarized in Table 4-17.

This monoclonal anti-HSA was chosen to be used in the albumin assay, as the polyclonal

antibody tested in 4.3.1.1, α-HSApoly, was not available to purchase.

Figure 4-24: Sensorgrams for α-HSA cross-reactivity with HSA, BSA and hIgG The left graph shows the sensorgrams for HSA, BSA and hIgG on the monoclonal antibody α-HSA, where HSA gave a high response. The right enlargement shows the weak binding of BSA on the same surface antibody. The bound BSA dissociated quickly from the surface after the injection was finished.

Analyte Conc. (μg/ml) Response (RU) % cross-reactivity Comment

HSAa 100 2184,5 100 % Set to 100 %

BSA 100 34,5 1,58 % Very low cross-reactivity

hIgG 100 8,1 0,37 % No cross-reactivity

Table 4-17: Cross-reactivity for α-HSA with HSA, BSA and hIgG The results from Figure 4-24 are summarized. The response for HSAa was set to 100 %. BSA displays cross-reactivity of 1.58 % which could be considered negligible.

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- CHAPTER 4 -

- 66 -

4.3.1.3. Human serum albumin preparations used as calibrator

During the evaluation of antibodies for the albumin concentration assay in section 4.3.1.1 and

4.3.1.2 an essentially fatty-acid free albumin preparation from Sigma, denoted HSAa, was used.

Later, as the assay was developed, it was found that the albumin in HSAa and in process samples

bind the monoclonal antibody α-HSA and the polyclonal antibody α-HSApoly differently. This

was found to give unreasonably high calculated concentrations of the albumin assay.

Therefore, three different HSA preparations were examined with the two antibodies α-HSA

and α-HSApoly. The preparations were HSAa; an essentially fatty-acid free albumin from Sigma,

HSAb; a fraction V preparation from Sigma, and finally the internally produced final albumin

preparation (sample #24b in section 4.3.4), purified essentially as described in Figure 2-1,

denoted HSAc.

The concentration of HSAa and HSAb were both established during the reconstitution from

lyophilized powder by weight of powder and final volume after added HBS-EP+ buffer to a

concentration of 2 mg/ml. The concentration of HSAc was determined by the average of biuret

measurements from the four final samples during the sterile filtration during purification.

When immobilizing α-HSA in flow-cell 1 to 9787 RU and α-HSApoly in flow-cell 2 to 10219

RU, and serially injecting the three preparations; the calibration curves were as in Figure 4-25.

For α-HSApoly, practically the same responses were observed for HSAa and HSAb while HSAc

gave higher responses. For α-HSA on the other hand, all three preparations showed different

responses with HSAa (green) lowest, HSAb (pink) middle and HSAc (red) highest response. The

albumin assay, using HSAa or HSAb as the calibrator with the monoclonal antibody α-HSA on

the surface, would result in an apparent higher calculated sample concentration.

Figure 4-25: Calibration curves α-HSA and α-HSApoly with three HSA preparations With α-HSApoly, HSAc gave a higher response while a difference could not be detected for HSAa and HSAb. This difference could be due to a difference in concentration due to a greater dilution of HSAc (from 200 mg/ml) than HSAa and HSAb (from 2 mg/ml). A significant difference was seen on α-HSA for all three preparations. HSAc gave a higher response over HSAb and HSAa. The monoclonal antibody binds the three preparations differently.

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- RESULTS -

- 67 -

To clarify if these differences were due to inadequate determination of concentrations, biuret

was used to control the concentration of preparation HSAa. Four 2.5-fold dilutions into water of

HSAa was analysed giving an average concentration of 1.98 mg/ml with CV 4.8 % (n=5). HSAa

was reconstituted in buffer HBS-EP+ which contains 0.5 mM EDTA that may interfere with the

biuret assay due to EDTA being a chelating agent that binds Ca2+ ions. That potential risk was

controlled by diluting a sample of known concentration into HBS-EP+ to 2.00 mg/ml, followed

by dilutions in water in the same manner as the HSAa sample. The biuret measurement of the

control gave 2.10 mg/ml with CV 9.4 % (n=10). Thus, this showed that the low concentration of

EDTA did not interfere with the biuret assay.

Experiments to determine the kinetic properties of the three preparations on the monoclonal

antibody were performed. As the results did not lead to any new interpretations or conclusions

the results are not shown.

From the above mentioned experiments, HSAc was chosen as the internal standard for the

albumin concentration assay in this study. This preparation of albumin was believed to be the

most appropriate to use in the assay as it will bind the antibody in the most identical manner to

all the process samples.

4.3.2. Assay development albumin concentration

The albumin concentration assay was very similar to the total IgG concentration assay

described in 4.1.2 and the lessons learned could be used for the development of the albumin

assay. The same standard curve concentrations were chosen, starting at 50 μg/ml HSAc with six

2.5-fold dilutions to approximately 0.5 μg/ml. The monoclonal α-HSA antibody had high activity

and regeneration performance and was thereby suitable to be used with a master standard curve

as described in 4.1.2. At least one start-up cycle was needed and one or two control samples were

appropriate.

4.3.2.1. Sample preparations

Samples were diluted to ensure that the sample concentration fall within the standard curve.

Samples expected not to contain albumin were diluted at a minimum 10 times to avoid pH,

buffer and NaCl effects. Samples in the process expected to contain albumin were diluted 1000

times. Samples close to the final product, after the final ultra-diafiltration with estimated

concentrations above 200 mg/ml were diluted 10000 times. These samples were diluted in two

steps in order to avoid pipetting of very small volumes into large volumes.

- CHAPTER 4 -

- 68 -


The procedure for the assay is the same as for total IgG in section 4.1.2.3 with some

deviations mentioned here. Immobilization was done with 15 μg/ml α-HSA for 7 minutes giving

approximately 11000 RU. Regeneration conditions were 30 seconds of glycine pH 2.0.

The quick, in-process analysis, for rapid analysis without an available master standard curve,

described for IgG in section 4.1.2.4 could most likely also be applied for albumin but was not

evaluated in this study.

A stability check was performed with 10 samples in 100 cycles each, four standard curves and

control samples. All samples gave reproducible results with all CV‟s below 1 %. Responses and

the CV for each sample are shown in Figure 4-26.

Figure 4-26: Stability check 1000 cycles albumin assay 10 samples with 100 cycles each with all CV‟s below 1 %. Exact CV‟s for each sample are shown in the figure.

4.3.3. International reference material calibration for albumin standard

The value transfer described in section 3.2.4 was applied on the albumin standard HSAc

(target material) utilised in the albumin concentration assay to calibrate the assay. The standard

used for albumin concentration assay (HSAc) was the final albumin product produced internally.

The concentration was determined by biuret to 200 mg/ml. The reconstitution of reference

material gave an albumin concentration of 37.56 mg/ml (CR), see Table 4-18.

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Calibration

Control

Sample

Startup

0.71% 0.63% 0.53% 0.86%

0.28% 0.39% 0.73%

0.15%

0.58% 0.60%

- RESULTS -

- 69 -

Reconstitution Reference Material

Concentration Albumin, mg/ml C'R 37,2000




Correction factor R 1,009795

Concentration Albumin, mg/ml CR 37,5644

Concentration Albumin, μg/ml CR 37564,37

Table 4-18: Reconstitution of reference material for albumin value transfer C'R is the certified concentration of albumin in ERM-DA470k/IFCC [14] and CR is the albumin concentration after reconstitution.

The standard curves for the three days of measuring are in Figure 4-27; each measurement

resulted in two curves. The reproducibility was impeccable with only two standard points from

day #1 deviating from the others. The dilution factor FT2 of the samples were plotted against the

relative concentration factors FR attained from interpolation of sample responses on standard

curve, as described in section 3.2.4. Linear regression with intercept set to zero, Figure 4-28, gave

the slopes 5.6151, 5.4957 and 5.7154 and for day #1, day #2 and day #3 of measurements

respectively.

Figure 4-27 (left): Standard curves for reference material albumin value transfer Impeccable reproducibility of the standard curves over the three days. Only two standard points from day #1 of measurements deviates slightly from the other days.

Figure 4-28 (right): Linear regressions for value transfer of albumin concentration Plotted results from: day #1 (), day #2 (), day #3 (▲). Linear regressions are the

blue lines with the equations: XY 5.61511 )9999.0( 2 R , XY 4957.52

)9998.0( 2 R and XY 5.71543 )0000.1( 2 R . The slope is equal to the

ratio of target material concentration and reference material concentration.

From these slopes the new albumin concentrations were calculated giving a mean value of

210.57 mg/ml, when the previous value was set to 200 mg/ml, shown in Table 4-19. The CV of

the value transfer was 1.96 % (n=6). The control sample had on average results of 99 %

compared to expected with CV of 3.29 %. The transfer factor for transformation of results from

measurements done prior to this calibration was calculated to 1.0528.

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Concentration %

HSA RM

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- CHAPTER 4 -

- 70 -

Results albumin value transfer Result conc. Concentration

Slope: Intercept: Control: in mg/ml Reference

Day #1 5,4957 0,0000 0,9517 206,4425 material 37,5644 mg/ml

Day #2 5,7154 0,0000 1,0090 214,6954

Day #3 5,6151 0,0000 1,0073 210,9277

Previous HSA conc. = 200 mg/ml

Mean: 5,61 0,989 210,5690 New HSA conc. = 210,57 mg/ml

Stand. Dev. 0,11 0,033 4,13 Transfer factor

CV %: 1,96 3,29 1,96 TF = 1,0528

Table 4-19: Results value transfer from reference material to HSAc Value transfer from the international reference material to HSAc resulted in a new albumin concentration of 210.57 mg/ml in the standard used in the study with a CV of 1.96 %. This also resulted in a transfer factor of 1.0528.

4.3.4. Results albumin assay on plasma-derived process samples

The albumin concentration assay described in section 4.3.2 with the protocol in Appendix E

was performed on samples from a lab-scale purification of albumin; performed essentially as

outlined in Figure 2-1. All samples were distributed from the starting plasma to the final albumin

product. One 96-well microplate was analysed with duplicate injections of all samples.

Measurements were done prior to the value transfer from international reference material

described above in section 4.3.3 and all concentrations were multiplied with the transfer factor

1.0528 to give the real concentrations presented here. Figure 4-29 shows the standard curve used

for the measurements of albumin process samples.

Figure 4-29: HSAc standard curve for albumin concentration assay The standard curve adjusted for new concentrations after value transfer from international reference material.

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ve

Re

sp

on

se

RU


HSA #299

Re

lati

ve

Re

spo

nse

(RU

)

- RESULTS -

- 71 -

The regeneration of α-HSA worked exceptionally well as seen on the baseline responses in

Figure 4-30. The only deviation is that of #13b, Alb DEAE Sepharose FF discarded fraction.

After this sample the baseline increased with 60 RU which was once again removed in the

following cycles. By disregarding #13b, the baseline only changes a total of 20 RU over 200

cycles which is extraordinarily good. The high and low control sample had on average 104.7 %

and 103.2 % compared to expected with CV of 2.5 % and 0.6 % respectively as seen in Table

4-20.

Concentration Calculated concentration

Compared to expected

μg/ml μg/ml CV %

High control 31,6 33,07 2,52 104,7 %

Low control 0,81 0,83 0,60 103,2 %

Table 4-20: Control samples albumin concentration assay The high and low control sample had good calculated concentration compared to expected, with CV‟s of 2.5 and 0.60 % respectively.

Figure 4-30: Baseline for albumin samples Exceptional regeneration of α-HSA with 30 seconds of glycine pH 2.0. Only sample #13b had incomplete regeneration resulting in a baseline increase of 60 RU. For all other 200 cycles the baseline only shifted 20 RU.

The results from 24 process samples are presented below in Table 4-21. Analysis was

performed in duplicates with three dilutions for each sample. Upon evaluation one dilution with

too high or too low concentration was always excluded leaving four determinations for each

sample. Results show very good reproducibility with CV‟s well below 1.0 % for all except two

samples.

Five samples (#18b-22b), after the final concentrating ultra-diafiltration step in the process,

gave unreasonably high concentrations. This was not verified but was believed to be due to

difficulties with the high dilution or that these samples were frozen and thawed prior to analysis,

while the two final samples (#23b-24b) were never frozen thus giving reasonable concentrations.

Because of these uncertainties the five overestimated samples will be excluded from further

interpretations.

47250

47350

47450

47550

47650

47750

0 50 100 150 200

Baseline: Sample

Ab

so

lute

resp

on

se -

baselin

e

RU

Cycle number

Calibration

Control

Sample

Startup

60 RU

Ab

sou

lte

Re

spo

nse

(RU

)

- CHAPTER 4 -

- 72 -

Results albumin concentration assay A

Biacore-assay

Biuret and SDS-PAGE B

# Purification step Fraction / Sample

Initial dilution

Conc. (mg/ml)

CV% (n=4)

Conc. (mg/ml)

1b Start Plasma pool 1000 35,4 0,29 32

2b Pre-treatment 1000 37,4 0,43 29

3b Filter Permeate 1000 33,5 0,68 31

4b FVIII S4FF FVIII fraction 10 0,01 0,46 0,0

5b FIX, Alb, IgG fraction 1000 16,8 0,11 15

6b FIX DEAE Alb,IgG fraction 1000 13,9 0,16 14

7b FIX fraction 10 0,01 0,55 0,0

8b Alb UF1 Retentate 1000 39,8 0,78 29

9b Alb SxG25 Alb, IgG fraction 1000 17,2 0,21 18

10b Euglobulin precipitation Supernatant 1000 20,3 0,53 19

11b Alb DEAE Alb fraction 1000 25,4 0,50 23

12b IgG fraction 10 0,01 0,99 0,0

13b Discarded fraction 10 0,03 19,5 0,3

14b Alb CM Alb fraction 1000 18,0 0,88 17

15b Alb UF2 Retentate 1000 82,9 0,49 72

16b Heat treatment Supernatant 1000 77,0 0,58 68

17b Sephacryl Alb fraction 1000 17,6 0,61 16

18b Alb UF3/DF Retentate 10000 259,1 C 0,22 214

19b Formulation 10000 242,9 C 0,58 215

20b Pasteurization 10000 257,5 C 0,42 224

21b Sterile filtration 1st filter 10000 295,6 C 0,50 210

22b 2nd filter 10000 217,7 C 0,68 197

23b 3rd filter 10000 207,9 0,72 195 24b Pasteurization Final product 10000 204,1 0,15 201

Table 4-21: Results albumin concentration assay A The samples were produced in lab-scale, essentially according to the plasma fractionation process outlined in Figure 2-1. B Concentration calculated from biuret total protein concentration multiplied with SDS-PAGE albumin purity in percentage, giving low reliability and sensitivity. C Results showing unreasonably high concentrations, probably due to dilution difficulties or the fact that samples #18b to #22b were frozen and thawed before analysis while #23b and #24b were never frozen, may be excluded from further interpretations. CV‟s were exceptionally good with all CV‟s, except for two samples, well below 1.0 %.

- RESULTS -

- 73 -

In Table 4-21 are also the concentrations obtained from multiplication of biuret total protein

concentration and SDS-PAGE purity of albumin. These results were compared with the results

from the Biacore albumin assay giving the correlation plotted in Figure 4-31. The two methods

correlates well with a slope from linear regression of 0.94 (R2 = 0.99). As discussed previously in

section 4.1.4 the biuret * SDS-PAGE method was highly uncertain.

Figure 4-31: Correlation between Biacore albumin results and biuret * SDS-PAGE results The concentrations from the albumin assay developed here were compared with concentrations calculated from total protein concentration from biuret measurements multiplied with an estimated purity of albumin from SDS-PAGE analysis. The concentrations correlate well with a slope of 0.94 with R2-value of 0.99 from linear regression with intercept set to zero. Samples #18b-22b were excluded as discussed previously.

In a similar manner as for the total IgG assay the discarded fraction sample from Alb DEAE

(#13b) expressed high unspecific binding for low dilutions. The repercussion was an increase in

the baseline for a few cycles and a very high CV of 19.5 %. Some samples that were only diluted

10 times and contained very low levels of NaCl demonstrated a negative bulk response in the

sensorgram, data not shown.

Due to the very good control samples and the good correlation with alternative methods the

results from the albumin concentration assay could be considered a successful analysis.

y = 0,9398x

R2 = 0,996

0

50

100

150

200

0 50 100 150 200

Biacore assay albumin (mg/ml)

Biu

ret

* S

DS

-PA

GE

(m

g/m

l)

- CHAPTER 4 -

- 74 -

4.4. Albumin specificity assay

An immunochemical Biacore assay to assess that the albumin in the samples was solely human

serum albumin and none was bovine serum albumin was evaluated. The aim was to have a highly

specific anti-bovine serum albumin antibody immobilized and samples injected serially over this

and α-HSA during analysis. Also a bovine serum albumin standard curve would be included.

4.4.1. Evaluations of reagents for albumin specificity assay

4.4.1.1. Immobilization and activity test of three monoclonal antibodies

Three monoclonal anti bovine serum albumin antibodies from Santa-Cruz Biotechnology

were evaluated for an assay intended to ensure the presence of solely human serum albumin and

none bovine serum albumin in the samples. Immobilization without prior pH scouting resulted

in levels of 10000 RU, 11000 RU and 3500 (pH 5.0) – 4500 (pH 4.5) RU for α-BSAa, α-BSAb

and α-BSAc respectively, for denotations see section 0. No interactions occurred with 120

seconds injections of HSA onto any of the flow-cells certifying no cross-reactivity with human

serum albumin (data not shown). An initial injection of 50 μg/ml reconstituted BSA from Sigma-

Aldrich only yielded in a 15 RU response on the higher immobilization of α-BSAc and no

response on the other flow-cells as shown in Figure 4-32.

Figure 4-32: Binding response of BSA on three anti-BSA monoclonal antibodies The purple curve represents α-BSAc immobilized to 4500 RU which showed a low response of about 15 RU when 50 μg/ml BSA was injected for 2 minutes. All other antibodies showed no interaction.

-10

-5

0

5

10

15

20

25

0 50 100 150 200 250

Adjusted sensorgram - BSA 50 µg/mlRU

Resp

on

se (

0 =

baselin

e)

sTime

α-BSAc (4500 RU)

Re

lati

ve

Re

spo

nse

(RU

)

- RESULTS -

- 75 -

4.4.1.2. Activity-test of anti-BSA with capture antibody

Immobilized α-mIgG (14126 RU) captured α-BSAa-c to levels between 1500 and 2000 RU,

with the set-up described in section 3.2.5 to ensure α-BSAa-c were not inactivated upon

immobilization. A second analyte injection of HSA was included to check for cross-reactivity.

Additionally, α-hIgG was captured with analyte hIgG as a control of the assay. Regeneration was

done with glycine pH 1.7. Results support those in section 4.4.1.1 showing a weak activity of α-

BSAc and no activity for the others. Also, no cross-reactivity with HSA was detected and the

control gave a positive result. The sensorgrams are displayed in Figure 4-33 and the results

summarized in Table 4-22. Due to the lack of activity of the anti-BSA antibodies and lack of time

to evaluate other antibodies; the specificity assay was excluded from the project.

Figure 4-33: Sensorgrams activity-test α-BSA with capture set-up The responses of the captured α-BSAa-c antibodies are seen in the left graph and the red curve is the control with α-hIgG and IgG. The enlargement to the right has a new baseline after the capture antibody injection. A very low binding can be seen for α-BSAc (purple).


Antibody RU BSA HSA

α-BSAa (2A3E6) 1852,7 4,3 3,9

α-BSAb (0.N.32) 1595,3 6,9 6

α-BSAc (BGN/D1) 2140,2 39,4 5,1

control (α-hIgG) 1456,9 (hIgG) 873,7 -----

Table 4-22: Results activity-test α-BSA with capture set-up All capture responses was fairly similar with 1500-2000 RU. No interactions were detected for α-BSAa-b but a weak binding for α-BSAc. No cross-reactivity was seen for either of the antibodies. The control with α-hIgG and IgG was positive.

-500

0

500

1000

1500

2000

2500

3000

0 100 200 300 400 500 600 700 800 900 1000


Re

sp

on

se (

0 =

cap

ture

_b

ase

lin

e)

sTim e

a-hIgG

anti-BSA (0.N.32)

anti-BSA (2A3E6)

anti-BSA (BGN/D1)

buffer

-10

0

10

20

30

40

50

60

200 300 400 500 600 700 800 900 1000


Re

sp

on

se

(0

= b

as

elin

e)

sTim e

BSA HSA

-500

0

500

1000

1500

2000

2500

3000

0 100 200 300 400 500 600 700 800 900 1000


Resp

on

se (

0 =

cap

ture

_b

aselin

e)

sTim e

a-hIgG

anti-BSA (0.N.32)

anti-BSA (2A3E6)

anti-BSA (BGN/D1)

buffer

Re

lati

ve

Re

spo

nse

(RU

)

Re

lati

ve

Re

spo

nse

(RU

)

- DISCUSSION -

- 77 -

Chapter 5

5Discussion

5.1. Total IgG concentration assay

Owing to the good performance of the already existing human antibody capture, the

immobilization and regeneration conditions were already optimized. The assay had a great

performance and only samples from two steps in the process showed interfering effects. First,

the discarded fraction eluted from the albumin DEAE Sephadex FF step demonstrated some

non-specific binding. Second, the discarded fraction containing solvent and detergent chemicals

from virus inactivation may affect the results, however needs further investigation.

5.2. IgG subclass distribution assay

After extensive antibody evaluation and assay optimization the IgG subclass distribution assay

also have good performance. The approach to use all flow-cells serially with the same standard

and sample injections was done with acceptable performance, but pushes the regeneration

performance to the limit on final IgG samples containing very high IgG1 and IgG2

concentrations and very low IgG3 and IgG4 concentrations, demanding a conditioning cycle

(dummy cycle). The low activity and regeneration properties with decreasing activity of all

antibodies except α-hIgG3 was limiting, but was compensated for by the trend calibration

software.

The antibody against human IgG3 had its epitope at the hinge region between Fc and Fab

domains on the immunoglobulin (Figure 2-2) [36]. As discussed and seen in Figure 2-3, IgG3 has

a very unique elongated hinge region with 62 amino acids compared to 12 in the others. This

could be the reason that α-hIgG3 had a much greater activity than all other subclass specific

antibodies, because the large unique hinge region was easier to specifically recognise and interact

- CHAPTER 5 -

- 78 -

with. The other subclass specific antibodies have their epitope on either the Fc domain or the

Fab domain [36]. The fact that IgG3 has a significantly lower biological half-life and higher

proteolytic activity could correspond with the greater regeneration performance of α-hIgG3, if

this means that IgG3 is less stable than the other subclasses.

5.3. Albumin concentration assay

The monoclonal anti-human serum albumin antibody evaluated for the albumin concentration

assay showed excellent activity and regeneration in Biacore. Due to differences in albumin

preparations used as calibrator the chosen calibrator for this study was the final product. An

alternative calibrator which binds the immobilized antibody in an identical manner as the samples

has to be found.

The discarded fraction eluted from the albumin DEAE Sephadex FF step demonstrated some

non-specific binding.

During analysis of the purified albumin, some of the highly concentrated final samples

showed variable and overestimated concentrations. This could have been due to freezing and

thawing of these concentrated samples or due to experimental variations in the very high dilution

(10000 times). The dilution scheme should therefore not contain any critical volumes below 10

μl; during the study, volumes of 2 μl was sometimes used. Non-critical dilutions, such as the

dilution of antibody for immobilization, may still be 1 to 2 μl in order to reduce reagent

consumption as this does not affect the assay performance.

5.4. Biacore assays, performance and comparison

All the developed Biacore assays had great performance. Compared to other evaluated

methods they were superior in terms of for example specificity, sensitivity, hands-on time, sample

and reagent consumption and for one assay also consumables cost, if excluding instrument costs.

Some of the compared measures on performance will be discussed below. The process samples

analysed in the study, were produced in lab-scale essentially according to the plasma fractionation

process outlined in Figure 2-1.

5.4.1. Specificity

The developed assays were immunochemical assays using specific monoclonal antibodies and

they were thereby highly specific. The antibodies were tested for cross-reactivity with other

relevant proteins and found not to cross-react. The traditional methods for the total IgG and

albumin concentration assays are a combination of total protein concentration by biuret and

- DISCUSSION -

- 79 -

purity by SDS-PAGE. This combination had a high inherent uncertainty as two methods with

low precision were merged together.

The subclass distribution assay on the other hand was compared with another

immunochemical assay, ELISA. This is a well-known, highly used method that also utilised

monoclonal antibodies for specificity against the IgG subclasses. The kit was intended for non-

purified plasma samples only and not optimized for process samples.

5.4.2. Sensitivity

As one aim for all assays was to reduce analysis and hands-on time as much as possible, the

sensitivity was secondary. Also, the concentrations in the samples typically range from 2 to 50

mg/ml for IgG and 15 to 220 mg/ml for albumin. That being said, the sensitivity for the total

IgG and albumin assays were still much higher than the biuret and SDS-PAGE method. The

lowest point on the standard curve was in both cases 0.51 μg/ml, with a minimum dilution of 10

times giving a lowest method detection limit of 5.1 μg/ml. If desired, sensitivity can easily be

increased further by prolonging injection time and decreasing lowest standard concentration.

The biuret assay, with a lowest detection limit of 500 μg/ml also had a very high variance. The

variance was further increased by the estimation of relative quantification by SDS-PAGE.

For the IgG subclass distribution assay different detection limits were obtained for each

subclass due to the different subclass concentrations in the calibrator and samples. The lowest

standard points were 2.8, 1.8, 0.4 and 0.2 μg/ml for the respective subclasses. As all samples were

diluted a minimum of 20 times, the lowest method detection limits were 56, 36, 8 and 4 μg/ml

for IgG1-4 respectively using the chosen conditions.

As previously stated, these limits could significantly be reduced by increased injection time

and using a lower standard concentration, but was not prioritised.

5.4.3. Resolution

From the fitted standard curve, the resolution of the total IgG assay was approximately 75 RU

per μg/ml. For the albumin assay it was approximately 40 RU per μg/ml. That is, for every 1

μg/ml increase in concentration the response increased with 75 RU and 40 RU for the total IgG

and albumin assay respectively. This was very high as the instrument can detect changes below 1

RU. Putting it differently, every 1 RU increase in response corresponded to a concentration

increase of 13 ng/ml and 25 ng/ml for the assays respectively.

For the fitted standard curves for the IgG subclass distribution assay the resolutions were,

very approximately, 5, 10, 40, 120 RU per μg/ml for IgG1-4 respectively. In other words, for

every 1 RU increase in response the concentration increased with 150, 100, 20 and 10 ng/ml for

IgG1-4 respectively.

All in all the resolution was more than sufficient for obtaining robust results.

- CHAPTER 5 -

- 80 -

5.4.4. Robustness

The assays had great robustness as samples from the chromatographic process were analysed

successfully with few exceptions. An individual evaluation of robustness was not performed due

to time constraints. Most of the potential matrix effects were removed by dilutions of all samples.

5.4.5. Hands-on and analysis time

Major benefits with the developed assays are the reduced hands-on time and analysis time.

Complete break-down of time consumption for the different analyses are shown in Appendix B.

For total IgG or albumin analysis of 22 samples, the hands-on time was reduced from

approximately 6 hours to 1 hour and the overall analysis time was reduced from 10 to 4.5 hours,

for Biacore assay compared to biuret and SDS-PAGE assay. Hands-on and analysis time for 1, 22

and 44 samples are displayed in Table 5-1. Immobilization was not included as it was only needed

at most once a week; it takes 30 minutes and can be performed unattended during sample

preparation. Results from one IgG or albumin sample was with Biacore readily available in 20

minutes with only 10 minutes total hands-on time where the same complete biuret and SDS-

PAGE analysis would take over 5 hours with almost 1.5 hour hands-on time. This is a great

advantage for in-process control analyses when a fast result is essential.

Time consumption (total IgG / albumin)

1 sample 1 sample 22 samples 44 samples

without st. curve with st. curve with st. curve with st. curve

Biacore (3 dilutions, 1 replicate)

Total hands-on 10 min 20 min 1 h 1,5 h

Total time 20 min 45 min 4,5 h 9 h

Biuret & SDS-PAGE (1 dilution, 2 replicates)

Total hands-on ----- 1,5 h 6 h 10 h

Total time ----- 5 h 10 h 16 h

Table 5-1: Hands-on and analysis time total IgG or albumin assay Approximated hands-on and analysis time for total IgG or albumin concentration and comparison between Biacore and biuret & SDS-PAGE assays. Only the Biacore assay is possible to execute without a standard curve. 22 samples were chosen due to the maximum of 11 samples per gel, and two gels per instrument on SDS-PAGE. A major contributor for the biuret & SDS-PAGE hands-on time were the evaluation of SDS-PAGE gels. Not included preparation of buffers and solutions for both assays and immobilization for Biacore. Immobilization (30 minutes unattended) is required at most once a week and can be performed during sample preparation.

Another comparison was made between the Biacore IgG subclass distribution assay and a

human IgG subclass ELISA kit. When using half the kit, one to five samples can be analysed in

duplicates and with the whole kit 17 samples in duplicates. Therefore the time consumption was

calculated for 1, 5 and 17 samples as seen in Table 5-2. For five samples, hands-on time was

decreased from 4 hours to 1 hour while overall analysis time was only decreased from 6.5 hours

to 6 hours. Whereas the overall unattended analysis time increases rapidly for the Biacore method

- DISCUSSION -

- 81 -

the hands-on time remains much lower as illustrated in Figure 5-1. Also worth to mention is that

the hands-on time for the ELISA was distributed over the whole analysis but for the Biacore

method the hands-on time was completed prior, hence analysis could be performed unattended

overnight.

Time consumption (IgG subclass distribution)

1 sample 5 samples 17 samples


(1 st. curve) (1 st. curve before and 1 after)

Total hands-on 40 min 1 h 1,5 h

Total time 2 h 6 h 15 h

ELISA (1 dilution, 2 replicates)

Total hands-on 2,5 h 4 h 6 h

Total time 5 h 6,5 h 8,5 h

Table 5-2: Hands-on and analysis time IgG subclass distribution assay Approximate hands-on (including evaluation) and total analysis time for IgG subclass concentration assay for Biacore and ELISA methods. 5 and 17 samples were chosen due to the maximum samples when using half or a whole ELISA kit. Not included preparation of buffers and solutions for all assays and immobilization for Biacore. Immobilization (2 hours unattended for four flowcells) is required at most once a week and can be performed during or prior to sample preparation.

Figure 5-1: Comparison hands-on and overall time IgG subclass With the Biacore method the hands-on time remain much lower despite number of samples while the overall analysis time increases but can be performed overnight. Both hands-on and analysis time increases with number of samples for ELISA and cannot be performed overnight.

0

2

4

6

8

10

12

14

16

0 10 20

No# samples

Tim

e, h

ou

rs

Biacore, hands-on

Biacore, total

ELISA, hands-on

ELISA, total

- CHAPTER 5 -

- 82 -

5.4.6. Consumables cost

The costs for the Biacore IgG subclass distribution assay was compared with the costs for the

same assay using an ELISA kit. In the Biacore method the sensor chip was the largest cost item.

But since the chip with antibodies immobilized can be used for a long time, it remains constant

for more samples. For ELISA, the kit itself was the most expensive. The kit was limited to a

maximum of 17 samples and more kits had to be accounted for when analysing larger number of

samples. Both for few or many samples, the Biacore method costs less with approximately 3600

SEK compared to 33000 SEK for 100 samples. This was not including instrument costs or the

labour cost for performing time consuming analysis.

17 samples 100 samples

Biacore Total

quantity Total cost

(SEK) Used

quantity Cost (SEK)

Used quantity

Cost (SEK)

Series S Sensor chip CM5 3 chips 4990 1 chip 1663 2 chips 3326 Amin coupling kit 50 sets 2470 1 set 49 2 sets 100 α-hIgG1 (500 μg/ml) 500 μl 1470 4 μl 12 8 μl 24

α-hIgG2 (1000 μg/ml) 1000 μl 3415 2 μl 7 4 μl 14

α-hIgG3 (1000 μg/ml) 1000 μl 3415 2 μl 7 4 μl 14

α-hIgG4 (1000 μg/ml) 1000 μl 3415 2 μl 7 4 μl 14 Calibrator (estimated maximum cost) 1000 μl 1000 10 μl 10 40 μl 20 Buffers, chemicals and other materials (estimated) 50 100 Total 1805 3612

17 samples 100 samples

ELISA Total

quantity Total cost

(SEK) Used

quantity Cost (SEK)

Used quantity

Cost (SEK)

Peliclass human IgG subclass ELISA kit

(1 kit = 17 samples) 5500 1 kit 5500 6 kits 33000

Total 5500 33000

Table 5-3: Comparison of costs for IgG subclass distribution assay The costs for 17 or 100 samples were calculated for Biacore and ELISA. The major expense for the Biacore assay was the sensor chip, but with an increased number of samples this cost item remains constant and always below the ELISA. For the ELISA method the kit was the only expense and was limited to 17 samples, therefore it became very expensive for an increased number of samples. Instrument costs and labour costs were excluded.

- RECOMMENDATIONS -

- 83 -

Chapter 6

6Recommendations

In the study, different calibrators were used for all assays. Instead, a common calibrator

similar to reference material containing known concentrations of all analysed plasma proteins

could be used. For use in QC or batch release, this new calibrator should be calibrated with the

developed assays against a reference material according to the value transfer protocol as

described in this report. A suitable example of a calibrator to be used is “Human serum protein

calibrator X0908” from DAKO, Denmark. This contains albumin, IgG with all subclasses

present and several others of the most abundant proteins in human plasma.

Additional assays could be developed in the same manner as the assays described here, to

quantify the major contaminants, such as transferrin, fibrinogen or haptoglobin. In the most

recent edition of European Pharmacopoeia 7.0, it was specified that the maximum content of

Immunoglobulin A (IgA) has to be indicated on an IgG product, determined by a suitable

immunochemical method [2]. Therefore, quantification of IgA could also be incorporated in the

Biacore methods.

As there are four flow-cells on one sensor chip, where two are occupied for IgG and albumin,

the two other could preferable contain one contaminant each. If possible, the common calibrator

discussed above could be used for all four assays.

The IgG subclass distribution assay works desirably but the antibody for human IgG2 has a

lower performance than the other three. Due to time and cost constraints additional antibodies

were not evaluated in this study. An additional interesting antibody for human IgG2 (clone

HP6002) was found and could be evaluated in a future study.

- CHAPTER 6 -

- 84 -

- REFERENCES -

- 85 -

Chapter 7

7References

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2. European Directorate for the Quality of Medicines & HealthCare (2011) European Pharmacopoeia, http://www.pheur.org

3. Matejtschuk, P., Dash, C. H., and Gascoigne, E. W. (2000) Production of human albumin solution: a continually developing colloid, Br J Anaesth 85(6): p. 887-895.

4. Orange, J. S., Hossny, E. M., Weiler, C. R., Ballow, M., Berger, M., Bonilla, F. A., Buckley, R., Chinen, J., El-Gamal, Y., Mazer, B. D., Nelson, R. P., Jr., Patel, D. D., Secord, E., Sorensen, R. U., Wasserman, R. L., and Cunningham-Rundles, C. (2006) Use of intravenous immunoglobulin in human disease: a review of evidence by members of the Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology, J Allergy Clin Immunol 117(4 Suppl): p. S525-553.

5. Burnouf, T., Padilla, A., Schaerer, C., Snape, T., and van Aken, W. G. (2005) WHO recommendations for the production, control and regulation of human plasma for fractionation, World Health Organization, Geneva.

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7. Meulenbroek, A. J., and Zeijlemaker, W. P. (1996) Human IgG Subclasses: Useful diagnostic markers for immunocompetence, http://www.xs4all.nl/~ednieuw/IgGsubclasses /subkl.htm (Retrieved in: January, 2011)

8. Horton, H. R., Moran, L. A., Ochs, R. S., Rawn, J. D., and Scrimgeour, K. G. (2002) Principles of biochemistry, 3rd ed., Pearson Education International, Upper Saddle River, NJ.

9. Nimmerjahn, F., and Ravetch, J. V. (2007) The antiinflammatory activity of IgG: the intravenous IgG paradox, J Exp Med 204(1): p. 11-15.

- CHAPTER 7 -

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10. Relkin, N. R., Szabo, P., Adamiak, B., Burgut, T., Monthe, C., Lent, R. W., Younkin, S., Younkin, L., Schiff, R., and Weksler, M. E. (2009) 18-Month study of intravenous immunoglobulin for treatment of mild Alzheimer disease, Neurobiol Aging 30(11): p. 1728-1736.

11. Sigma-Aldrich. (2011) Human Albumin, http://www.sigmaaldrich.com/life-science/metabolomics /enzyme-explorer/enzyme-reagents/human-albumin.html (Retrieved in: January, 2011)

12. Blirup-Jensen, S. (2001) Protein standardization III: Method optimization basic principles for quantitative determination of human serum proteins on automated instruments based on turbidimetry or nephelometry, Clin Chem Lab Med 39(11): p. 1098-1109.

13. Whicher, J., Baudner, S., Bienvenu, J., Blirup-Jensen, S., Carlstrom, A., Dati, F., Johnson, A. M., Ritchie, R. F., Svendsen, P. J., and Milford-Ward, A. (1996) New initiatives in the standardization of protein measurements, Pure & Appl. Chem. 68(10): p. 1851-1856.

14. Zegers, I., Schreiber, W., Sheldon, J., Blirup-Jensen, S., Muñoz, A., Merlini, G., Itoh, Y., Johnson, A. M., Trapmann, S., Emons, H., and Schimmel, H. (2008) Certification of proteins in the human serum. Certified reference Material ERM-DA470k/IFCC, EUR Report 23431 EN European Community, Luxembourg.

15. Emons, H., Linsinger, T., and Gawlik, B. M. (2004) Reference materials: terminology and use. Can't one see the forest for the trees?, Trends Anal. Chem. 23(6): p. 442-449.

16. Blirup-Jensen, S., Johnson, A. M., and Larsen, M. (2001) Protein standardization IV: Value transfer procedure for the assignment of serum protein values from a reference preparation to a target material, Clin Chem Lab Med 39(11): p. 1110-1122.

17. Blirup-Jensen, S., Johnson, A. M., and Larsen, M. (2008) Protein standardization V: value transfer. A practical protocol for the assignment of serum protein values from a Reference Material to a Target Material, Clin Chem Lab Med 46(10): p. 1470-1479.

18. Rawlins, P. (2010) Current trends in label-free technologies, Drug Discovery World (Fall 2010): p. 17-26.

19. Cooper, M. A. (2006) Current biosensor technologies in drug discovery, Drug Discovery World (Summer 2006): p. 68-82.

20. Liedberg, B., Nylander, C., and Lundstrom, I. (1983) Surface plasmon resonance for gas detection and biosensing, Sensors Actuators 4: p. 299-304.

21. Schasfoort, R., and Tudos, A. (2008) Handbook of Surface Plasmon Resonance, The Royal Society of Chemistry, RSC Publishing, Cambridge.

22. Jonsson, U., Fagerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Lofas, S., Persson, B., Roos, H., Ronnberg, I., and et al. (1991) Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology, Biotechniques 11(5): p. 620-627.

23. Fagerstam, L. G., Frostell-Karlsson, A., Karlsson, R., Persson, B., and Ronnberg, I. (1992) Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis, J Chromatogr 597(1-2): p. 397-410.

24. GE Healthcare. (2007) Technology Note 23: Lable-free interaction analysis in real-time using surface plasmon resonance, http://www.biacore.com.

25. Johnsson, B., Löfås, S., Lindquist, G., Edström, A., Müller Hillgren, RM., Hansson, A. (1995) Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies, J Mol Recognit 8(1-2), p. 125-131

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26. Johnsson, B., Löfås, S., Lindquist, G. (1991) Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors, Anal Biochem 198(2), p. 268-277

27. GE Healthcare. (2007) Application Note 48: Validation of a concentration assay using Biacore C, http://www.biacore.com.

28. Crosson, C., Thomas, D., and Rossi, C. (2010) Quantification of immunoglobulin g in bovine and caprine milk using a surface plasmon resonance-based immunosensor, J Agric Food Chem 58(6): p. 3259-3264.

29. Jiang, X., Waterland, M., Blackwell, L., Wu, Y., Jayasundera, K. P., and Partridge, A. (2009) Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing, Steroids 74(10-11): p. 819-824.

30. Fonfria, E. S., Vilarino, N., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2008) Feasibility of using a surface plasmon resonance-based biosensor to detect and quantify yessotoxin, Anal Chim Acta 617(1-2): p. 167-170.

31. Karlsson, R., Fagerstam, L., Nilshans, H., and Persson, B. (1993) Analysis of active antibody concentration. Separation of affinity and concentration parameters, J Immunol Methods 166(1): p. 75-84.

32. Pol, E. (2010) The importance of correct protein concentration for kinetics and affinity determination in structure-function analysis, J Vis Exp(37).

33. Richalet-Secordel, P. M., Rauffer-Bruyere, N., Christensen, L. L., Ofenloch-Haehnle, B., Seidel, C., and Van Regenmortel, M. H. (1997) Concentration measurement of unpurified proteins using biosensor technology under conditions of partial mass transport limitation, Anal Biochem 249(2): p. 165-173.

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35. Schauer, U., Stemberg, F., Rieger, C. H., Borte, M., Schubert, S., Riedel, F., Herz, U., Renz, H., Wick, M., Carr-Smith, H. D., Bradwell, A. R., and Herzog, W. (2003) IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the binding site reagents, Clin Chem 49(11): p. 1924-1929.

36. Reimer, C. B., Phillips, D. J., Aloisio, C. H., Moore, D. D., Galland, G. G., Wells, T. W., Black, C. M., and McDougal, J. S. (1984) Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes, Hybridoma 3(3): p. 263-275.

- 89 -

Appendix A Regeneration scouting α-hIgG2

Regeneration scouting α-hIgG2 with 10mM Glycine pH 3.0-1.5

Regeneration scouting α-hIgG2 with NaCl 0.5-5M / Ethylene glycol 50-100%

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

48600

48800

49000

49200

49400

49600

49800

50000

50200

50400

50600

RU RU

Cycle

Re

sp

on

se B

ase

line


0

50

100

150

200

250

300

350

0 5 10 15 20 25 30 35

48000

48500

49000

49500

50000

50500

51000

51500

52000

RU RU

Cycle

Resp

on

se B

aselin

e


0

50

100

150

200

250

300

350

0 3 6 9 12 15 18 21 24 27

47800

48000

48200

48400

48600

48800

49000

49200

49400

49600

RU RU

Cycle

Re

sp

on

se B

ase

line


0

50

100

150

200

250

300

350

0 3 6 9 12 15 18 21 24 27

47800

48000

48200

48400

48600

48800

49000

49200

49400

49600

RU RU

Cycle

Re

sp

on

se B

ase

line


0,5 M 1 M 3 M 4 M 5 M NaCl NaCl NaCl NaCl NaCl

50% 75% 100% Ethylene glycol

Glycine Glycine Glycine Glycine pH 3.0 pH 2.5 pH 2.0 pH 1.5

- 90 -

Regeneration scouting α-hIgG2 with MgCl2 1-4M

Regeneration scouting α-hIgG2 with SDS 0.02-0.5%

50

100

150

200

250

300

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

48000

48500

49000

49500

50000

50500

51000

RU RU

Cycle

Resp

on

se B

aselin

e


0

50

100

150

200

250

300

350

0 2,3 4,6 6,9 9,2 11,5 13,8 16,1 18,4 20,7 23

45500

46000

46500

47000

47500

48000

48500

49000

RU RU

Cycle

Resp

on

se B

aselin

e


0

50

100

150

200

250

300

350

0 3 6 9 12 15 18 21 24 27

47800

48000

48200

48400

48600

48800

49000

49200

49400

49600

RU RU

Cycle

Re

sp

on

se B

ase

line


0

50

100

150

200

250

300

350

0 3 6 9 12 15 18 21 24 27

47800

48000

48200

48400

48600

48800

49000

49200

49400

49600

RU RU

Cycle

Re

sp

on

se B

ase

line


1 M 2 M 3 M MgCl2 MgCl2 MgCl2 4 M

MgCl2

0,02% 0,05% 0,1% SDS SDS SDS 0,2% 0,5% SDS SDS

- 91 -

Appendix B Hands-on and analysis time

Time consumption (total IgG / albumin)

1 sample 1 sample 22 samples 44 samples

(without st. curve) (with standard curves)


hands-on time, min 5 min 10 min 35 min 60 min

cycle time, sec 195 s 195 s 195 s 195 s

no of cycles 3 9 72 138

total analysis, min 10 min 29 min 215 min 429 min

total analysis, hour 0,2 h 0,5 h 4 h 7 h

Evaluation time, min 5 min 5 min 20 min 30 min

Total, hour 0,3 h 0,7 h 4,5 h 8,7 h

Biuret (1 dilution, 2 replicates) (high+low st. curves)

no of plates ----- 1 2 2

hands-on time, min ----- 10 min 45 min 60 min

incubation time, min ----- 30 min 30 min 30 min

plate read, min ----- 10 min 10 min 10 min

total analysis, min ----- 40 min 50 min 50 min

Evaluation time, min ----- 10 min 30 min 50 min

Total, min ----- 60 min 125 min 160 min

Total, hour ----- 1,0 h 2,1 h 2,7 h

SDS-PAGE (1 dilution, 1 replicates)

no of gels ----- 1 2 4

hands-on time, min ----- 30 min 90 min 180 min

no of runs ----- 1 1 2

run time, min ----- 70 min 70 min 140 min

Staining, hour ----- 60 min 60 min 60 min

Destaining, hour ----- 60 min 60 min 60 min

Evaluation time, hour ----- 0,5 h 3 h 6 h

Total, hour ----- 4,2 h 7,7 h 13,3 h

Time consumption (IgG subclass distribution)

1 sample 5 samples 17 samples


(1 st. curve) (1 st. curve before and 1 after)

hands-on time, min 20 min 35 min 45 min

cycle time, sec 500 s 500 s 500 s

no of cycles 11 37 97

total analysis, hour 1,5 h 5,1 h 13,5 h

Evaluation time, min 20 min 30 min 30 min

Total, hour 2,2 h 6,2 h 14,7 h

ELISA (1 dilution, 2 replicates)

no of plates 1 1 2

hands-on time, hour 2,2 h 3,3 h 4,8 h

incubation time, hour 2,5 h 2,5 h 2,5 h

Evaluation time, hour 0,5 h 0,8 h 1,0 h

Total, hour 5,2 h 6,5 h 8,3 h

- 92 -

Appendix C Protocol total IgG concentration assay

A) Immobilization (~10000 RU)

1) Dilute anti-human IgG antibody in Sodium Acetate pH 5.0 to 25μg/ml (5μl + 95μl) from

Human Antibody Capture Kit (0.5mg/ml, BR-1008-39, GEHC)

2) Immobilization wizard (use Amine coupling kit, BR-1000-50, GEHC)

Method: Amine

Contact time: 360 s

Flow rate: 5 μl/min

B) Biacore-method: Concentration assay

1) General settings

1 Hz, Single detection, 25°C temperature, (10°C for long runs), unit: μg/ml

2) Cycle type “concentration”

Sample: i. Type: low sample consumption ii. Contact time: 20s, dissociation time: 0s, flow rate: 20 μl/min

Regeneration: i. 3M MgCl2 ii. Contact time: 30s, flow rate: 30 μl/min iii. Predip

3) Assay-steps

Startup: 1-3 replicates

Sample: 1 replicate

Calibration (if not using master standard curve): repeat within „Sample‟, e.g. before / after / every 400 cycles

Control: repeat within „Sample‟, e.g. every 36 cycles

4) Variable settings

Startup: e.g. IgG 8 μg/ml (from calibration dilution)

Sample: define sample solution and dilution at run time

Calibration: IgG 0.51, 1.28, 3.2, 8, 20, 50 μg/ml

Control: e.g. IgG 1.28, 20 μg/ml (from calibration dilution)

5) Setup run

Enter all samples with three dilutions, (e.g. 800, 400, 200X)

Set rack positions accordingly

- 93 -

C) Sample and standard preparations

1) Quick vortex of all samples

2) Spin samples with visible precipitation (13g for 1 minute)

3) Initial sample dilution (appr. 200-1000μl final volume) in HBS-EP+

1000X: Expected IgG concentration > 10mg/ml

200X: Expected to contain IgG

20X: Not expected to contain IgG (i.e. to detect losses)

4) Two additional two-fold dilutions of samples in HBS-EP+ For several samples (up to 32), e.g. transfer 200μl to 96-well microplate, add 100μl HBS-EP+ to subsequent wells with multi-pipette and dilute 100μl + 100μl with multi-pipette.

5) Dilute standard to 50μg/ml and five 2.5-fold dilutions in HBS-EP+ (50, 20, 8, 3.2, 1.28, 0.51 μg/ml)

6) Dispense standards, controls and regeneration solution 3M MgCl2 in vials with caps in Reagent Rack 2

D) Evaluation

1) If using master standard curve, import with “Append result file…”

2) Concentration analysis / using calibration

3) Use two of the three dilutions for each sample concentration evaluation.

Add: 200 μl, 8 samples

100 μl HBS-EP+ 100 μl HBS-EP+

200 μl, 8 samples 100 μl HBS-EP+ 100 μl HBS-EP+



Transfer & mix 100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

1 2 3 4 5 6 7 8

9 … …

- 94 -

Appendix D Protocol IgG subclass distribution assay

A) Immobilization (8000-11000 RU)

1) Dilute anti-human IgG subclass antibodies in Sodium Acetate pH 5.0 to 20μg/ml:

anti-human IgG1 (4μl + 96μl), (0.5mg/ml, MH1015, Invitrogen)

anti-human IgG2 (2μl + 98μl), (1mg/ml, MC005, Immunkemi/The binding site)

anti-human IgG3 (2μl + 98μl) , (1mg/ml, MC006, Immunkemi/The binding site)

anti-human IgG4 (2μl + 98μl) , (1mg/ml, MC009, Immunkemi/The binding site)


Method: Amine

Fc1: α-hIgG1, Fc2: α-hIgG2, Fc3: α-hIgG3, Fc4: α-hIgG4

Contact time: Fc1: 660s, Fc2: 600s, Fc3: 420s, Fc4: 420s



1) General settings

1 Hz, Multi detection, 25°C temperature, (10°C for long runs), unit: % or μg/ml


Sample: i. Type: low sample consumption ii. Contact time: 120s, dissociation time: 0s, flow rate: 5 μl/min iii. Flow path: 1,2,3,4

Regeneration 1: i. 12.5 mM NaOH ii. Contact time: 60s, flow rate: 10 μl/min iii. Flow path: 1,2 iv. Predip

Regeneration 2: i. 12.5 mM NaOH ii. Contact time: 30s, flow rate: 10 μl/min iii. Flow path: 1,2,3,4 iv. Predip v. Stabilization period: 60s

3) Assay-steps

Startup: 10 replicates (if new surface)

Sample: 1 replicate

Calibration: repeat within „Sample‟, e.g. before / after / every 48 cycles

Dummy-cycle: repeat within „Sample‟, every 5 cycles

Control: repeat within „Sample‟, e.g. Before / every 15 cycles

- 95 -


Startup: e.g. IgGSc 320X dilution (from calibration dilution)


Calibration: IgGSc 1280, 640, 320, 160, 80, 40X dilution,

i. Insert relative concentration (%) [=100/dilution],

0.078, 0.156, 0.3125, 0.625, 1.25, 2.5 % evaluation D) or

ii. Insert one IgG subclass concentration (μg/ml),

e.g. IgG1: 2.8, 5.7, 11.3, 22.6, 45.2, 90.4 evaluation E)

Dummy-cycle (conditioning cycle): HBS-EP+

Control: e.g. IgGSc 320X dilution (from calibration dilution)

5) Setup run

Enter all samples with five dilutions (e.g. 1280, 640, 320, 160, 80)






160X: Expected IgG concentration ~40-50mg/ml

80X: Expected to contain IgG

20X: Late in process, expected IgG concentration <5mg/ml

10X: Not expected to contain IgG (i.e. to detect losses)

4) Four additional two-fold dilutions of samples in HBS-EP+ For several samples (up to 18), e.g. transfer 200μl to 96-well microplate, add 100μl HBS-EP+ to subsequent wells with multi-pipette and dilute 100μl + 100μl with multi-pipette.

5) Dilute standard 40X and five 2-fold

dilutions in HBS-EP+, (40X1280X)

6) Dispense standards, controls and regeneration solution 12.5mM NaOH in vials with caps in Reagent Rack 2

D) Evaluation, using relative concentration (%) (T200 evaluation software)

1) Concentration analysis / Using calibration / Use calibration trends

2) Select flow cell for IgG1-4

3) Use three of the five dilutions for each sample concentration evaluation.

4) Multiply all obtained concentrations (%) with specified IgGSc concentration in the

standard (μg/ml), divided by 100. Example: measured concentration = 0.5% IgG1 in standard = 3620μg/ml Measured IgG1 concentration = 0.5*3620/100=18.1 μg/ml

5) Restart from 2) for each IgGSc


100 μl HBS-EP+ 100 μl HBS-EP+ 100 μl HBS-EP+ 100 μl HBS-EP+

200 μl, 8 samples 100 μl HBS-EP+ 100 μl HBS-EP+ 100 μl HBS-EP+ 100 μl HBS-EP+

2 samples . + controls .

Transfer & mix 100 μl

100 μl 100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

1 2 3 4 5 6 7 8

9 … …

- 96 -

E) Evaluation, using concentration (μg/ml) (T200 evaluation software)

1) Create one evaluation file for each IgGSc

2) Tools / Keyword table

Change Conc (μg/ml) to the specific IgG subclass concentration

(e.g. for IgG1 2.8, 5.7, 11.3, 22.6, 45.2, 90.4 μg/ml for standard dilutions 1280X to 40X)

3) Concentration analysis / Using calibration / Use calibration trends

4) Select flow cell for same IgG subclass as in 2)

5) Use three of the five dilutions for each sample concentration evaluation.

6) Restart from 1) for each IgGSc

- 97 -

Appendix E Protocol albumin concentration assay

A) Immobilization (~11000 RU)

1) Dilute monoclonal anti-human serum albumin antibody in Sodium Acetate pH 5.0 to

~16 μg/ml (1μl + 65μl) (1.07mg/ml, Ab399, Abcam)


Method: Amine

Contact time: 420 s



1) General settings

1 Hz, Single detection, 25°C temperature, (10°C for long runs), unit: μg/ml


Sample: i. Type: low sample consumption ii. Contact time: 20s, dissociation time: 0s, flow rate: 10 μl/min

Regeneration: i. Glycine pH 2.0 ii. Contact time: 30s, flow rate: 20 μl/min iii. Predip

3) Assay-steps

Startup: 1-3 replicates

Sample: 1 replicate

Calibration: (if not using master standard curve): repeat within „Sample‟, e.g. before / after / every 400 cycles

Control: repeat within „Sample‟, e.g. every 36 cycles


Startup: e.g. HSA 8 μg/ml (from calibration dilution)


Calibration: HSA 0.51, 1.28, 3.2, 8, 20, 50 μg/ml

Control: e.g. HSA 1.28, 20 μg/ml (from calibration dilution)

5) Setup run

Enter all samples with three dilutions, (e.g. 2000, 1000, 500X)


- 98 -





10000X: Expected HSA concentration > 200mg/ml (avoid pipetting small

volumes: dilute twice, e.g. 10μl + 990μl 10μl + 990μl)

500X: Expected to contain HSA

20X: Not expected to contain HSA (i.e. to detect losses)

4) Two additional two-fold dilutions of samples in HBS-EP+ For several samples (up to 32), e.g. transfer 200μl to 96-well microplate, add 100μl HBS-EP+ to subsequent wells with multi-pipette and dilute 100μl + 100μl with multi-pipette.

5) Dilute standard to 50μg/ml and five 2.5-fold dilutions in HBS-EP+ (50, 20, 8, 3.2, 1.28, 0.51 μg/ml)

6) Dispense standards, controls and regeneration solution glycine pH2.0 in vials with caps in Reagent Rack 2

D) Evaluation

1) If using master standard curve, import with “Append result file…”

2) Concentration analysis / using calibration

3) Use two of the three dilutions for each sample concentration evaluation.


100 μl HBS-EP+ 100 μl HBS-EP+




Transfer & mix

100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

100 μl

1 2 3 4 5 6 7 8

9 … …

Documents

New SPR based assays for plasma protein titer determination. - DiVA