Co-Expression and Co-Purification Enable Manufacturing of a Six-Monoclonal Antibody Botulinum Antitoxin Cocktail

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Abstract
A highly potent antitoxin for botulinum neurotoxin (BoNT) serotypes A and B has been developed that comprises three monoclonal antibodies (mAbs) targeting BoNT/A and three targeting BoNT/B. These oligoclonal antibody combinations neutralize toxin by simultaneously binding non-overlapping epitopes, thereby promoting rapid toxin clearance. All six mAbs use the same human Fc and framework and have been individually manufactured using the same expression platform and purification process. To minimize the time and labor required to produce the divalent antitoxin, we tested a co-expression and co-purification strategy for the three mAbs per serotype. The mAbs were expressed in CHO-K1 cells, and the media were optimized for co-expression in 10 L bioreactors. Chromatographic co-purification consisted of Protein A capture, followed by strong anion exchange chromatography in flow-through mode and cation-exchange chromatography in bind-elute mode. Co-expression experiments demonstrated that expression of the three anti-BoNT/A antibodies remained within approximately ±30% of the optimal equimolar ratio, whereas the anti-BoNT/B antibodies showed greater variability. Downstream purification steps achieved recoveries greater than 95% per chromatographic step, resulting in overall process yields of approximately 63–75%. This strategy provided sufficient purity of all six mAbs while largely preserving their relative ratios. These results demonstrate the feasibility of producing oligoclonal antitoxin antibodies using co-expression and shared purification strategies. Such approaches may simplify the manufacturing of antibody cocktails while maintaining product quality and biological activity.
Funding Information
  • U.S. Department of War (W911SR22D0009)
  • Defense Threat Reduction Agency (W15QKN-16-9-1002)

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