Avoiding antibody aggregation during processing: Establishing hold times

Abstract

Aggregation of biotech products used therapeutically, such as antibodies, can contribute to potential immunogenicity of the product. Charge-based heterogeneities may also impact the safety and/or efficacy of a therapeutic. In this study, an approach based on empirical modeling and least squares regression is suggested for establishing hold times for process intermediates during production of monoclonal antibody (Mab) therapeutics. Two immunoglobulins were analyzed with respect to aggregation and charge heterogeneity in buffer conditions that are typically used during downstream processing of Mab products. Size exclusion chromatography, ion exchange chromatography (IEC), and circular dichroism were used. We found that aggregation primarily occurs at pH 3 (buffers used in affinity chromatography) and is higher in citrate buffer compared to acetate and glycine buffers. Aggregation is minimal in buffers used in anion exchange chromatography (Tris-HCl buffer at pH 7.2 and 8) and in cation exchange chromatography (citrate buffer at pH 6, acetate buffer at pH 6, and phosphate buffer at pH 6.5 and 7.5). The behavior is opposite in the case of charged heterogeneities (basic and acidic variants) as measured by IEC. The product is more susceptible to degradation at high pH than at low pH. The data presented here demonstrate that product stability can be a significant issue within the routinely used manufacturing conditions. We suggest that the approach presented needs to be adopted by all manufacturers to ensure product stability during processing.

Aggregation Kinetics for IgG1-Based Monoclonal Antibody Therapeutics

“…4d). One of the possible explanations for this can be the rearrangement of aromatic amino acids (tryptophan and tyrosine) in the citrate buffer (both in presence and absence of salt) environment (23). This behavior is consistent with the effect of buffer species on stability of mAb therapeutics that have been reported in the literature (23).…”

“…4a) (23). It should be noted that there are differences observed in the final % aggregate in comparison to what has been previously observed and reported (23). The reason for this is that though the starting material in the two cases was from the same product but it came from different batches and had different % aggregate at time t = 0.…”

“…It is observed that at low pH, the MRE values become positive as the time progresses and this signifies that there are structural changes in the protein molecule which lead to conformational loss and subsequently aggregate formation (Fig. 4a) (23). It should be noted that there are differences observed in the final % aggregate in comparison to what has been previously observed and reported (23).…”

“…Table II presents a summary of the aggregation behavior that was seen under various storage conditions. It was seen that aggregation is high at low pH and worsens with addition of salt and increase in temperature (23). Aggregation was minimal under most conditions at high pH.…”

“…In a previously published study, we have elucidated the importance of establishing hold times during mAbs processing (6,23). In this paper, we focus on evaluation of the aggregation kinetics for immunoglobulin (IgG1)-based mAb therapeutics.…”