Versatile LC–MS-Based Workflow with Robust 0.1 ppm Sensitivity for Identifying Residual HCPs in Biotherapeutic Products

Abstract

Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography−mass spectrometry/mass spectrometry (LC−MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC−MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10-to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying >85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.

Technical advancement and practical considerations of LC-MS/MS-based methods for host cell protein identification and quantitation to support process development

“… 12 In parallel, both lower and high sensitivity LC-MS/MS methods can be applied to the characterization of upstream bioprocesses and knockout cell lines. 22–25 In addition to the increased sensitivity, the industry has also improved overall method robustness, by demonstrating recovery of specific high-risk proteins 7 , 28 , 29 or set of multiple HCP standards. 30 With robustness shown for a single method, the applications of LC-MS/MS methods are expanding into HCP comparability assessment 10 , 17 and good manufacturing practice (GMP) release testing.…”

“…HCP monitoring occurs at various stages of the biotherapeutics production workflow, starting from early upstream processing and including in-process pools and drug products from preclinical, clinical stages and the final commercialized product. 6 , 7 If a high-risk HCP is detected during the bioprocess in drug substance, a toxicity risk control strategy should be applied to monitor and remove HCP to ensure patient safety. 1 Most antibodies and many therapeutic proteins are generated from Chinese hamster ovary (CHO) or E. coli cells so the majority of analytical methods are developed for HCPs of biotherapeutics from these two types of cell lines.…”

“… 12 This has driven the need for increasingly sensitive methods for HCP detection and quantitation, due to the wide dynamic range of the HCP population, with more than six orders of magnitude difference in different process pools. 7 , 13 …”

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

“…This is accomplished by omitting the standard digest's reduction and denaturation steps prior to digestion and then precipitating the mAb after digestion. Several mAb depletion (HCP enrichment) strategies have been derived from the native digest method to enhance coverage of HCPs in purified mAb solutions 23–27 . However, a concern with all these techniques is that HCP peptides may be lost during the mAb removal step, leading to uncertainty as to which digest method is better suited for the analysis of incompletely purified mAb solutions, such as HCCF.…”

diaPASEF Enables High-throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development

“…Alternatively, native digestion selectively digests proteolytically more labile HCPs over mAbs. 11 In addition, offline fractionation 12,13 , long gradient LC 14 , and ion mobility spectrometry 15,16 improve separation between HCP– and therapeutic protein–derived peptides. However, these strategies typically involve somewhat laborious sample preparation and/or require long instrument time (> 2 h / sample), which considerably limits sample throughput.…”