Characterization and implications of host‐cell protein aggregates in biopharmaceutical processing

Abstract

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.

Analytical characterization of host‐cell‐protein‐rich aggregates in monoclonal antibody solutions

“…The large aggregates appear to have been implicated in the persistence of more than a thousand HCPs through protein A chromatography, as hypothesized in prior work. 46 This is more clearly shown in Figure 4d, which compares the HCP overlap between the common samples derived from HCCF and PAVIN. Roughly 80% of HCPs in PAVIN aggregates were also identified in the corresponding HCCF aggregates, which is about double what was observed previously.…”

“…This may be the result of CHO lysosomal protein degradation as well as protease activity in free solution. 46,62 The mean HCP mass in the SPF fractions is significantly lower than that in the HCCF aggregates, but the effect size is small. Similarly, a comparison of isoelectric point distributions does not reveal significant differences aside from a slightly higher mean pI in the HCCF SPF 2 fraction than in the other HCCF samples (Supplementary Figure S3).…”

“…Roughly 80% of HCPs in PAVIN aggregates were also identified in the corresponding HCCF aggregates, which is about double what was observed previously. 46 Comparisons of HCP biophysical properties across SEC fractions reveal significant but relatively small effect sizes. About 10% of the HCPs identified in the SPFs have unphysically large molecular masses of >100 kDa, which is presumably due to protein fragmentation (Supplementary Figure S2).…”

“…Separate work recently confirmed this idea, showing that hundreds of HCPs may be present in aggregates in mAb solutions. 45,46 The biophysical basis of this phenomenon was attributed at least in part to the association between mAbs and HCPs involved in the unfolded protein response. Oh et al 46 suggested that the resulting aggregates may directly mediate the persistence of many HCPs through protein A chromatography, but analysis of the extent of this phenomenon was limited by the different digest methods that were used to analyze HCCF and protein A eluate samples.…”

“…Although this observational study is limited in scope to one mAb product, we expect the trends to be transferrable to other mAb solutions that contain HMW impurities given similar observations that were made in a recent study of several mAb products. 46…”