Kinetic Analysis of Antibodies from Different Cultured Media - A label-free platform is evaluated to establish an analytical method for assessing the effects of media condition on the quality and acti

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Kinetic Analysis of Antibodies from Different Cultured Media
A label-free platform is evaluated to establish an analytical method for assessing the effects of media condition on the quality and activity of cultured antibodies.


BioPharm International
pp. 48-51, 55


Figure 3a: Example of typical raw data sensorgram collected from protein/ligand binding experiment. Sensorgram is the kinetic profile of 25 g/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.
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 106 cells/mL by day 4, while the BalanCD CHO Growth A culture increased steadily and peaked above 4 x 106 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 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.
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).


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

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,

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," http://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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