BioPharm International www.biopharminternational.com July 2013

David Ho* is a scientist, Tom Fletcher is director, cell culture, and

Jessie H.T. Ni, PhD, is chief scientific officer, all at R&D Department, Irvine Scientific,

2511 Daimler Street, Santa Ana, California, USA.

*To whom all correspondance should be addressed, dho@irvinesci.com.

peer-revieweD

Article submitted: March 15, 2012. Article accepted: May 13, 2013.

Antibody Quality Determination

T radit ional ly, the culture growth and biologic titer production have been used exclusively to evaluate the

performances of cell lines and the culture environment, such as media development optimization. Several analysis approaches have evolved to characterize the quality of the biologics produced, (e.g., ant ibodies have been of great interest due to fast growth of biosimilar and biobetter business). These approaches focus on determining the antibody quality from information related to the antibody purity, charge variant, glycan, and kinetics. In this study, the authors eva lu ate t he O c te t QKe p lat-for m to e s t abl i sh a n a na ly t i -c a l m e t h o d t o a s s e s s m e d i a condit ion ef fect on the qual ity and activity of cultured antibodies by measuring the affinity and kinetics of monoclonal antibodies (mAbs) produced from a hybridoma cell line cultured in two different types of medium. With this method, the intermolecular binding interactions

between the antibodies and targeted protein can be measured to identify an approach to the activity of interest, such as an in v ivo therapeutic response. The qualitative measurement, in addition to cell growth and titer production, can be useful to further develop and select essential media components to improve product functionality to a cell line.

MATeRIAlS AnD MeThoDS Growth culture and titer. A selected mouse hybridoma cell line, HFN 7.1, producing antihuman fibronectin mAb was thawed and cultured in two Irvine Scientific media, IS MAB-CD (catalog no. 91104) and BalanCD CHO Growth A (catalog no. 91128), for a seven-day period at 37 ºC with 5% CO

2

(1). Both culture media, IS MAB-CD a nd Ba la nCD CHO Grow t h A , contain a combination of common media components, although BalanCD CHO Grow th A i s a ver sat i le , chemica l ly def ined, ser um-f ree med ium, more robust t ha n I S MAB - CD in amino ac ids, v ita-

abstractthe Octet QKe platform is evaluated to establish an analytical method for assessing the effects of media condition on the quality and activity of cultured antibodies. the affinity and kinetics of monoclonal antibodies produced from a hybridoma cell line cultured in two different types of medium were measured. With this method, the authors were able to measure the intermolecular binding interactions between the antibodies and targeted protein. the qualitative measurement, in addition to cell growth and titer production, can be useful to further develop and select essential media components to improve product functionality to a cell line.

Kinetic Analysis of Antibodies from Different

Cultured MediaDavid Ho, Tom Fletcher, and Jessie H.T. Ni

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Antibody Quality Determination

mins, and trace metal components. The cell density and viability were counted at selected time points in the seven-day cultivation with a Beckman Coulter Vi-Cell Counter. Titer quantitation was performed on days 6 and 7 with the Octet QKe, using Protein A biosensors.

Kinetic analysis. The Octet QKe platform uses a proprietary biolayer interferometry (BLI) technology to perform real-time, labe l - f ree qua nt it at ion a nd k inet ic characterization of biomolecular interac-tions of antibodies, proteins, peptides, and small molecules. The mouse antihuman f ibronec t in a nt ibod ies f rom day 7 cultured media were aliquoted and stored for kinetic analysis to the matched paired target protein, human fibronectin (catalog no . 1918 - F N - 0 2 M ), R& D Sy s t e m s .

The Octet QKe platform uses chemical biosensors to immobilize either the ligand (antibody) or protein (f ibronectin) to the tip of the biosensors to interact with their respective pair for binding measure-ments conducted in a 96-well plate format. Two t y pes of biosensor approaches, anti-mouse IgG Fc capture (AMC) and streptavidin (SA) biosensors were used to immobilize the antibody and fibronectin, r e sp e c t ive ly. F ib rone c t i n wa s p r e -pared for immobilization by undergoing buffer exchange and biotinylation with the Pierce biotinylation kit (2). Ligand/protein loading concentrat ions were optimized to their specified biosensors to measure binding activity with respective protein/ligand to obtain aff inity and kinetic measurements.

ReSUlTS Growth culture and titer. From culture monitor-ing, the growth of the HFN 7.1 cell line was higher in IS MAB-CD reaching up to 5 x 106 cells/mL by day 4, while the B a l a nC D C H O Gr ow t h A c u l t u r e increased steadi ly and peaked above 4 x 106 cell/mL on day 6 (see Figure 1a). Viabil ity decreased at day 4 for IS MAB-CD culture and around day 6 for BalanCD CHO Growth A culture (see Figure 1b). The cumulat ive growth of the IS MAB - CD was comparable to BalanCD CHO Growth A. Titer produc-tion was observed to be slightly higher by 50 mg/L in BalanCD CHO Growth A compared to IS MAB-CD (see Figure 2). A

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Figure 1: Cell density and viability analyzed at selected time points in seven-day cultivation to profile HFN 7.1 cell line culture in selected Irvine Scientific medium. (a) Viable cell density graph of HFN 7.1 cultures. (b) Percent viability graph of HFN 7.1 cultures.

IS MAB-CD

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Figure 2: Antibody production of HFN 7.1 cultures. Titer production taken at day 6 (green) and day 7 (blue) of HFN 7.1 cultures in Irvine Scientific medium.

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Antibody Quality Determination

Kinetic analysis. For kinetics analysis, day 7 culture samples were collected and used to simulate typical practice of end of culture analysis. The cell-culture fluid samples from titer measurements were used “as is” accompanying a serial dilu-tion gradient with kinetics buffer into a well plate column for analysis prepara-tion. An attractive appeal to the Octet platform is the ability to use cell culture f luid with no further sample process-ing step (e.g., purification) in which the external particulate components in the surrounding sample solution have mini-mal effect on the signal based on the BLI technology compared with other kinetic analysis platforms. Figure 3a is an exam-ple of a typical generated kinetic step sen-sorgram for either ligand/protein loading to specified biosensor with on-rate (asso-ciation) and off-rate (dissociation) binding to the complementing protein/ligand. The raw data were processed to fit a 1:1 binding model (red lines) to extract kinetics and aff inity measurements (see Figure 3b). The measured kinetic rates and affinities of AMC biosensors immobilized with antibodies cultured in IS MAB-CD and BalanCD CHO Growth A to bind with Fibronectin resulted with on-rates of 1.54 x 106 M-1s -1 and 1.47 x 106 M-1s -1, off-rates of 8.71 x 10-4 s-1 and 9.05 x 10-4 s-1, and affinity (equilibrium dissociation constants) of 0.57 nM and 0.62 nM. The measured kinetic rates and affinities of SA biosensors immobilized with biotinylated f ibronect in to bind with ant ibodies cultured in IS MAB-CD and BalanCD CHO Growth A medium resulted with on-rates of 2 . 58 x 10 5 M -1s -1 a nd 2 .0 0 x 10 5 M -1s -1, o f f - r a te s o f 1 .0 0 x 10 - 3 s -1 a nd 1. 21 x 10 - 3 s -1, a nd a f f i n i t y (equilibrium dissociation constants) of 3.9 nM and 6.1 nM. These results are summar ized in Tables I and II . From an article with relevance to label-free detection, using optical waveguide light-mode spectroscopy (OWLS) to analyze f ibronectin layers with mAb reported aff inity measurements within similar order of magnitude range to the AMC approach (3).

Figure 3a: Example of typical raw data sensorgram collected from protein/ligand binding experiment. Sensorgram is the kinetic profile of 25 μg/mL biotinylated fibronectin immobilized on streptavidin (SA) biosensors measuring on-rates (association) and off-rates (dissociation) of antibodies from IS MAB-CD with gradient dilutions.

Figure 3b: Example of processed data analyzed to 1:1 fitting. Selected concentration curves were fit to 1:1 binding model displayed in red for affinity and kinetic measurements.

Raw data (Sensor location)2.40

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Loading sample ID: Bt-Fibronectin; SampleID: mAb1; Sensor Info: -by Color; Conc. (nM)

Table I: Summary of kinetics (kdissociation, kassociation) and affinity (equilibrium dissociation, KD) results for anti-mouse IgG Fc capture (AMC) experiment.

Irvine Scientific (IS) media kdis [1/s] ka [1/Ms] KD [M]

IS MAB-CD 8.71E-04 1.54E+06 0.57 nM

BalanCD CHO Growth A 9.05E-04 1.47E+06 0.62 nM

Table II: Summary of kinetics (kdissociation, kassociation) and affinity (equilibrium dissociation, KD) results for streptavidin (SA) experiment.

Irvine Scientific (IS) media kdis [1/s] ka [1/Ms] KD [M]

IS MAB-CD 1.00E-03 2.58E+05 3.9 nM

BalanCD CHO Growth A 1.21E-03 2.00E+05 6.1 nM

BioPharm International www.biopharminternational.com July 2013

Antibody Quality Determination

DISCUSSIon AnD ConClUSIonA hybridoma cell line was cultured in two different medium types to evaluate the quality of antibodies produced to establish an analyti-cal method using the Octet QKe platform. The kinetics and affinity measurements obtained by the two biosensor approaches for ligand/protein binding interac-tions demonstrated an order of magnitude difference that might be related to orientation of ligand/protein binding to the biosensor. Between the two approaches, the authors believe that the AMC bio-sensor approach would be more closely representative of the in vivo environment for binding function between antibody and protein, in which, the Fc portion of the anti-body was immobilized on the bio-sensor tip to align and expose the

fragment antigen binding (Fab) portion for binding with fibronec-tin. In the SA biosensor approach, biotinylation of fibronectin might have affected the protein’s dimer configuration environment to result in higher affinity measurements observed due to the protein being three times larger than the antibody.

From the results of the two medium cultured in the hybridoma cell line, the growth profile and titer production was significantly improved with BalanCD CHO Growth A media, while the affin-ity of the antibodies produced in the IS MAB-CD displayed slightly higher affinity trends in both approaches. Dependent on the established parameter goals for a cell line, the overall growth pro-file, titer production, and kinetic analysis information can be useful

as determinant factors for choos-ing the proper culture medium to evaluate the performance of a specific cell line. Further studies should investigate the mechanistic pathway between the antibody and targeted protein, as well as additional culture medium condition types to improve design of experiments for identifying the critical media com-ponents to produce the desired anti-body quality.

RefeRenCeS1. R.C. Schoen et al., Hybridoma 1 (2)

99-108 (1982). 2. Fortebio website, “Technical Note

28: Biotinylation of Protein for Immobilization onto Streptavidin Biosensors,” www.fortebio.com, accessed May 28, 2013.

3. C. Wittmer and P.R. Van Tassel, Colloids and Surfaces B: Biointerfaces 41 (2-3) 103-109 (2005). ◆

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