yOur PATH TO SuCCESS

Antibody Arrays for Biosimilar Conformational

Comparability Analysis

Array Bridge provides ELISA based analysis for Host Cell

Proteins from CHO (Chinese Hamster Ovary ) and E. coli

host. The development and production of the ELISA kits

are under cGMP to ensure uninterrupted product supply

and consistent quality. Host Cell Proteins are proteins and

their derivatives from the hosts used for biologics

development and production. Higher levels of HCP will

pose risks in several areas.

First, HCPs from CHO and E. coli could be recognized by

the human immune system as no self molecules, eliciting

an immune reaction. Depending on the level and composi-

tion of the HCPs, the reaction could range from none and

mild reaction to severe reactions. Secondly, some HCPs

could induce the production of antibodies. If the HCP has

a homologous counterpart in the human body, these

antibodies could interfere with the physiological function

of the protein through cross-reactivity. Thirdly, HCPs

themselves could act as agonist or antagonist to interfere

with the human normal metabolism. Because of these

reasons, in biologics development, significant resource is

allocated during process development to reduce HCP

levels in the biologics product.

PrOCESS-rELATED IMPurITy AnALySIS

3-D COnFOrMATIOnAL COMPArABILITy AnALySIS

Protein Conformational Array ELISA provides a

systematic, sensitive and robust comparability testing

for biologics (therapeutic proteins) at molecular level.

An array of polyclonal antibodies was designed systemati-

cally covering the whole biologics sequence and the assay

is in an easy-to-use ELISA format, these Protein Confor-

mational Array ELISAs (PCA ELISA) can provide valuable

information on the 3-D structure and heterogeneity of

biologics, and can be used at many stages and aspects

of biologics development including cell line selection,

process development, formulation development and

product release testing.

It is known that the clinical and biological properties of a

biologics are the results of their basic properties such as

amino acid sequence and three-dimensional structure, as

well as the production, purification, formulation and storage

conditions. One of the major challenges in biologics develop-

ment is protein immunogenicity, unwanted immunogenicity

could lead to reduced or loss of drug efficacy, altered

pharmacokinetics (PK), general immune and hypersensitivity

reaction, and neutralization of the natural counterpart in

the human body. Multiple studies have demonstrated that

protein conformation stability is closely related to its

immunogenicity. One recent study indicated that a protein

has a threshold of conformational stability to prevent the

immunogenicity of foreign proteins. Another strong indica-

tion that protein conformation is closely related to its

immunogenicity is through the study of protein aggregation.

Multiple studies showed that protein aggregation is a major

source of immunogenicity.

In the recently published document for biosimilar

development by the Food and Drug Administration

(Guidance for Industry, Quality Considerations in

Demonstrating Biosimilarity to a reference Protein

Product, FDA, February 2012), FDA recommends

“Extensive, robust comparative physicochemical and

functional studies should be performed to evaluate

whether the proposed biosimilar product and the

reference product are highly similar. A meaningful

assessment as to whether the proposed biosimilar

product is highly similar to the reference product

depends on, among other things, the capabilities of

available state-of-the-art analytical assays to

assess, for example, the molecular weight of the

protein, complexity of the protein (higher order

structure and post-translational modification), degree

of heterogeneity, functional properties, impurity

profile, and the degradation profiles denoting stability.”

The FDA guidance

further stated that

“The three dimensional

conformation of a

protein is an important

factor in its biological

function. Protein

generally exhibit

complex three-dimen-

sional conformations

(tertiary structure

and, in some cases,

quaternary structure) due to their large size and the

rotational characteristics of protein alpha carbons.

The resulting flexibility enables dynamic, but subtle,

changes in protein conformation over time, some of which

may be absolutely required for functional activity.”

“... at the same time, a protein’s three-dimensional

conformation can often be difficult to define precisely

using current physiochemical analytical technology.”

Because of the close relation between protein conforma-

tion and its immunogenicity, several analytical techniques

and bioassays have been used to probe conformational

comparability in biologics. For example, protein intrinsic

fluorescence, analytical ultracentrifugation, gel filtration,

light scattering and bioassays have all been employed for

protein conformational analysis. However these approach-

es have their respective limitations, generally they lack

the desired sensitivity, coverage and throughput to

provide the information about protein 3-D structure.

In the case of monoclonal antibody biologics, Bioassays

developed based on target-antibody recognition will

detect some changes in the CDr (complementarity

determining region) regions, but can’t measure changes

in the rest of the biologics molecule.

The Protein Conformational Array developed specifically

toward monoclonal antibody drugs could provide a

sensitive, systematic and efficient way to measure protein

conformational comparability. The protein conformational

array antibodies are developed from the specific sequence

of each monoclonal antibody drug; about 30 different

antibodies were developed to provide a systematic

coverage of the molecule. Studies using marketed

monoclonal antibody drugs indicated that these confor-

mational arrays can provide detailed information about

the molecule and detect changes that may not be detected

by the aforementioned techniques including bioassays.

PrOTEIn COnFOrMATIOnAL ArrAyS, A nEW APPrOACH FOr BIOLOGICS THrEE–DIMEnSIOnAL STruCTurE COMPArABILITy AnALySIS

ApplicAtion — 1

COMPArISOn BETWEEn MArKETED MOnOCLOnAL AnTIBODy DruG AnD BIOSIMILAr CAnDIDATE.

ApplicAtion 2

FOrMuLATIOn DEvELOPMEnT

Ab10Ab9

Ab8

Ab7

Ab6

2.5Ab

5

Ab4

Ab3

Ab2Ab1

Ab11

Ab12

Ab13

Ab14

Ab15

Ab16

Ab17

Ab18

Ab19

Ab20

Ab22

Ab24

Ab23

Ab25

Ab21

Ab26

Ab27

Ab28

Ab29

Ab30

Reference Lot 2Reference Lot 1 Reference Lot 3 Biosimilar

2

1.5

1

0

0.5

OD 4

50 n

m

Comparison of Three Lots of Innovator Molecule and a Biosimilar Candidate

Ab10Ab9

Ab8

Ab7

Ab6

2.5

Ab5

Ab4

Ab3

Ab2Ab1

Ab11

Ab12

Ab13

Ab14

Ab15

Ab16

Ab17

Ab18

Ab19

Ab20

Ab22

Ab24

Ab23

Ab25

Ab21

Ab26

Ab27

Ab28

Ab29

Ab30

OD 4

50 n

m

Application of Conformational Array to Formulation Development

Control Pi Buffer, O ion Pi Buffer+400mM NaCL Histidine Buffer

2

1.5

1

0

0.5

Array Bridge provides products and services that

address two important areas in the development of

biologics: biosimilar drug comparability analysis and

impurity analysis.

During biosimilar development, biologics comparability

is critical to the successful development of the process

and product. From cell line selection to process develop-

ment, from formulation development to change control,

comparability is closely related to the molecule’s safety

and efficacy. Array Bridge has developed ‘antibody arrays’

to measure biosimilar drug comparability at the molecular

level, providing a sensitive, systematic and robust mea-

surement of biosimilar conformational comparability.

This antibody array-based technology can be used at all

stages of biosimilar development, from cell line selection and

process development to clinical testing and product release.

For impurity analysis, Array Bridge provides ELISA kits and

reagents for Host Cell Protein quantitation and Western

Blot analysis. All the products are manufactured under

cGMP to ensure quality and consistency. In the near future,

Array Bridge will also provide Q-PCr-based residual DnA

quantitation kits for CHO and E. coli-derived biologics and

an ELISA kit for residual Protein A quantitation.

In addition to these products, Array Bridge also provides

services to the biotech industry. If you need to analyze your

samples for impurities (Host Cell Proteins, residual DnA or

Protein A) or for biosimilar conformational comparability,

Array Bridge has scientists with experience working under

cGMP and ICH guidelines to deliver regulatory-ready results

and documentation. Further, we can help you with ELISA

method qualification or validation. The company also

provides consultation for the development of an effective

impurity analysis strategy, enabling your program(s) to

meet requirements from regulatory agencies such as FDA

and EMA.

Array Bridge provides value to our customers through

high quality products and services, and we help you

develop biologics including biosimilars successfully.

ABOuT ArrAy BrIDGE

ArB-1125

SErvICES

ArrAy Bridge provides services in severAl AreAs

1. Biosimilar conformational comparability analysis.

2. CHO and E. coli Host Cell Protein quantitation, method development, qualification

or validation.

3. Western Blot analysis of HCPs in CHO or E. coli-derived biologics using 1-D and 2-D

gel electrophoresis and 1-D and 2-D Western Blot.

4. Development of customer-based biosimilar conformational comparability analysis

ELISA and HCP ELISA.

5. Providing consultation on the Host Cell Protein strategy for a specific project

or platform.

www.arraybridge.comphone: 636-284-4212.

email: support@arraybridge.com

4320 Forest Park Avenue. Ste. 303

St. Louis, MO 63108

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