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

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.


PHOTO CREDIT: NICHOLAS RIGG/GETTY IMAGES
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.


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)
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% CO2 (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 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.
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.


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