Characterization of a cathepsin D protease from CHO cell‐free medium and mitigation of its impact on the stability of a recombinant therapeutic protein

Abstract

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:120-129, 2018.

Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance

“…A more likely cause for the increase in aggregates and fragments could be the presence of proteases—it has previously been reported that proteases are produced during late stage cell culture and can cause product fragmentation. Cathepsin D in particular is known to co‐purify and cleave the antibody product . Accumulation of fragments could also lead to the formation of aggregates.…”

Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

“…CHO cells are widely used for production of recombinant glycoprotein therapeutics due to their high productivity (1-10 g per liter), genetic stability and ability to grow in large-scale suspension culture [199][200][201] in serum-free medium. However, many recombinant proteins, including monoclonal antibodies, antibody fusion proteins and IFN-γ, are partially degraded or proteolyzed by endogenous CHO cell proteases during the cell culture or recovery process [202][203][204][205][206][207]. Novel approaches to solve proteolysis and enhance glycosylation for production of HIV envelope proteins as vaccine candidates are being explored [208].…”

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

“…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).…”

“…Although high clearance of HCPs are often achieved as determined by enzyme-linked immunosorbent assay (ELISA; X. 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). Thus, it is important to identify and characterize these HCPs and to design efficient separation processes for their removal.…”

“…A PLBL2-specific ELISA was developed to track its clearance throughout the chromatographic steps and was used to guide the design of separation processes for its removal (B. Tran et al, 2016). 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). Several studies have been carried out to confirm that mAb-HCP interactions were responsible for their lack of removal during protein A capture and subsequent polishing steps (Chollangi et al, 2015;Doneanu et al, 2012;Soderquist, Trumbo, Hart, Zhang, & Flynn, 2015;B.…”