Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product

Abstract

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co-purification of trace levels of 'hitchhiker' HCPs during the manufacturing process. Purified mAb-1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed-onset particles containing mAb-1 and its heavy chain C-terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb-1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb-1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP-mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb-1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb-1 product.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

“…Cathepsins in the culture supernatants have been reported as enzymes that break down proteins23242526. In particular, Ctsd cleaves mAbs, generating C-terminal heavy chain fragments12. In this study, five different cathepsins (Ctsa, Ctsb, Ctsd, Ctsl, and Ctsz) were identified, with their concentrations gradually increasing during the cultures (see Supplementary Table S7).…”

“…These HCPs were themselves classified into various clusters. For example, cathepsin D (Ctsd) generates C-terminal heavy chain fragments by cleaving mAbs12. Ctsd and other cathepsin proteases including Ctsa, Ctsb, Ctsl, and Ctsz were associated with lysosome and classified in cluster 2 for both batch and fed-batch cultures.…”

“…In particular, proteases789 and glycosidases1011 that accumulate in culture medium negatively affect the quality of mAbs in recombinant CHO (rCHO) cell cultures. In addition, although the concentrations of HCPs in cell culture harvests are reduced to acceptable levels after a series of purification steps, certain HCPs escape an entire purification process and remain in the final mAb drug substance at levels that affect product quality and stability1213. Therefore, it is paramount to characterize and quantify HCPs in fed-batch cultures to ensure an optimal mAb quality and perform targeted removal of HCPs during the purification steps.…”

Investigation of cathepsin D–mAb interactions using a combined experimental and computational tool set

“…Wang, Hunter, & Mozier, 2009), there may still be specific HCPs in the final product that can cause degradation of the drug product or elicit an immune response in patients (Bee et al, 2015;Fischer et al, 2017;Gao et al, 2011;Lim et al, 2017;Robert et al, 2009). Proteomic studies have shown that certain HCPs such as phospholipase B, lipoprotein lipase, and some serine proteases can persist at detectable levels through multiple chromatographic processes (Bee et al, 2015;B. The carry-over of HCPs through all the polishing steps to the final drug product can be attributed to either coelution with the product during chromatographic steps or product association via nonspecific interactions (Gaitatzis, Sisodiya, & Sander, 2012;Shukla & Hinckley, 2008;Yuk et al, 2015).…”

“…PLBL2 was identified to interact with different mAbs and to "hitchhike" through the entire process, resulting in degradation of some of the formulation components. Cathepsin D was found to cause mAb degradation in the formulated drug product (Bee et al, 2015) and various strategies have been used to remove residual cathepsin D, either by optimal washes during protein A capture or by thermal inactivation of the protease (Lim et al, 2017). Cathepsin D was found to cause mAb degradation in the formulated drug product (Bee et al, 2015) and various strategies have been used to remove residual cathepsin D, either by optimal washes during protein A capture or by thermal inactivation of the protease (Lim et al, 2017).…”