Strategy to mitigate aggregation during Protein A chromatography and low pH virus inactivation for a nivolumab biosimilar candidate.

Protein A chromatography represents the most prevalent methodology for the capture of monoclonal antibodies. The use of a low pH elution buffer from Protein A has been observed to contribute to product aggregation, particularly in the case of IgG4 antibodies, such as nivolumab. This paper presents a well-defined strategy for addressing this issue. Initial experiments were conducted at scale-down Protein A affinity chromatography to evaluate the use of glycine-HCL and sodium citrate as elution buffers at pH values of 3.25, 3.5, and 3.75. Subsequently, a scale-down screening was conducted to assess the efficacy of various additives in Protein A elution. These included 10 % (w/v) mannitol, 50 mM histidine, 50 mM sucrose, 10 % (v/v) sorbitol, 50 mM arginine, 50 mM trehalose, 0.02 % (v/v) polysorbate 80, 1.5 M urea, and 1 M MgCl. The three most stabilizing additives were evaluated at the laboratory scale, and the one that demonstrated the greatest ability to maintain the minimum high molecular weight aggregate over time was selected. Lastly, the selected additive was subjected to testing at elevated IgG concentrations during purification. Nivolumab exhibits a markedly pH-dependent propensity for aggregation, and the relative efficacy of glycine-HCL and sodium citrate in mitigating anti-PD1 aggregation within the pH range of 3.25 to 3.75 is subject to variation. The use of buffer 100 mM sodium citrate, pH 3.5 was found to be beneficial. All additives evaluated contribute to reducing nivolumab aggregation, albeit in different ways and to varying degrees of effectiveness. Elution buffer with mannitol, polysorbate 80, or MgCl₂ resulted in a monomer control ratio of approximately twice that observed in the absence of additives. However, the stabilizing role of mannitol was confirmed to be particularly significant, as the ratio of aggregation formed at a low pH was reduced to ≤ 2 % from 15 % in all evaluated scales and at different protein concentrations, while maintaining high biological activity.