Kinetic Analysis of Antibodies from Different Cultured Media

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BioPharm International, BioPharm International-07-01-2013, Volume 26, Issue 7

An analytical method assesses the effects of media condition on cultured antibodies.

ABSTRACT

The 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.

Traditionally, 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., antibodies 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 evaluate the Octet QKe platform to establish an analytical method to assess media condition effect on the quality 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 vivo 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.

PHOTO CREDIT: NICHOLAS RIGG/GETTY IMAGES

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 medium, IS MAB-CD (catalog no. 91104) and BalanCD CHO Growth A (catalog no. 91128), for a seven-day period at 37 °C with 5% CO

2

(1). Both culture media, IS MAB-CD and BalanCD CHO Growth A, contain a combination of common media components, although BalanCD CHO Growth A is a versatile, chemically defined, serum-free medium, more robust than IS MAB-CD in amino acids, vitamins, 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.

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. (ALL FIGURES ARE COURTESY OF THE AUTHORS)

Kinetic analysis. The Octet QKe platform uses a proprietary biolayer interferometry (BLI) technology to perform real-time, label-free quantitation and kinetic characterization of biomolecular interactions of antibodies, proteins, peptides, and small molecules. The mouse antihuman fibronectin antibodies from day 7 cultured media were aliquot and stored for kinetic analysis to the matched paired target protein, human fibronectin (catalog no. 1918-FN-02M), R&D Systems. The Octet QKe platform uses chemical biosensors to immobilize either the ligand (antibody) or protein (fibronectin) to the tip of the biosensors to interact with their respective pair for binding measurements conducted in a 96-well plate format. Two types of biosensor approaches, anti-mouse IgG Fc capture (AMC) and streptavidin (SA) biosensors were used to immobilize the antibody and fibronectin, respectively. Fibronectin was prepared for immobilization by undergoing buffer exchange and biotinylation with the Pierce biotinylation kit (2). Ligand/protein loading concentrations were optimized to their specified biosensors to measure binding activity with respective protein/ligand to obtain affinity and kinetic measurements.

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

RESULTS

Growth culture and titer.

From culture monitoring, the growth of the HFN 7.1 cell line was higher in IS MAB-CD reaching up to 5 x 10

6

cells/mL by day 4, while the BalanCD CHO Growth A culture increased steadily and peaked above 4 x 10

6

cell/mL on day 6 (see Figure 1a). Viability decreased at day 4 for IS MAB-CD culture and around day 6 for BalanCD CHO Growth A culture (see

Figure 1b

). The cumulative growth of the IS MAB-CD was comparable to BalanCD CHO Growth A. Titer production was observed to be slightly higher by 50 mg/L in BalanCD CHO Growth A compared to IS MAB-CD (see

Figure 2

).

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.

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 dilution gradient with kinetics buffer into a well plate column for analysis preparation. An attractive appeal to the Octet platform is the ability to use cell culture fluid with no further sample processing step (e.g., purification) in which the external particulate components in the surrounding sample solution have minimal effect on the signal based on the BLI technology compared with other kinetic analysis platforms. Figure 3a is an example of a typical generated kinetic step sensorgram for either ligand/protein loading to specified biosensor with on-rate (association) 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 affinity 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-1 s-1 and 1.47 x 106 M-1 s-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 fibronectin to bind with antibodies cultured in IS MAB-CD and BalanCD CHO Growth A medium resulted with on-rates of 2.58 x 105 M-1 s-1 and 2.00 x 105 M-1 s-1, off-rates of 1.00 x 10-3 s-1 and 1.21 x 10-3 s-1, and affinity (equilibrium dissociation constants) of 3.9 nM and 6.1 nM. These results are summarized in Tables I and II. From an article with relevance to label-free detection, using optical waveguide lightmode spectroscopy (OWLS) to analyze fibronectin layers with mAb reported affinity measurements within similar order of magnitude range to the AMC approach (3).

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.

DISCUSSION AND CONCLUSION

A hybridoma cell line was cultured in two different medium types to evaluate the quality of antibodies produced to establish an analytical method using the Octet QKe platform. The kinetics and affinity measurements obtained by the two biosensor approaches for ligand/protein binding interactions 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 biosensor 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 antibody was immobilized on the biosensor tip to align and expose the fragment antigen binding (Fab) portion for binding with fibronectin. 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.

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

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 affinity 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 profile, titer production, and kinetic analysis information can be useful as determinant factors for choosing 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 components to produce the desired antibody quality.

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

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

REFERENCES

1. 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).