host-cell-protein-analysis-using-enrichment-beads-coupled-with

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

Regeneron Pharmaceuticals Inc.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cutoff (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP with low molecular weight (11 kDa and 17 kDa) at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment.

Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product as excipients to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to shortened drug shelf lives and PS-degrading activity in drug products must be minimized. Identification of lipases that degrade PS could lead to better process control in drug manufacturing. In 2016, phospholipase B-like 2 (PLBD2) was proposed as a residual host cell protein responsible for degrading PS20 in a drug formulation. We have carried out a series of studies to verify the role of PLBD2 in degrading polysorbates in drug products purified from recombinant Chinese Hamster Ovary (CHO) cells. Genetic knock-out and immuno-depletion results showed that when PLBD2 was removed or depleted, the degradation of PS20 or PS80 was neither diminished nor reduced. In addition, a quantitative analysis of PLBD2 and PS20 degradation in multiple formulated mAb products did not establish a correlation between the amount of PLBD2 and the level of PS20 degradation. Collectively these results suggest that PLBD2 is not the primary cause of polysorbate degradation in formulated drug products purified using standard Protein A and ion exchange chromatography.

Putative Phospholipase B-Like 2 is Not Responsible for Polysorbate Degradation in Monoclonal Antibody Drug Products.

Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to particulate formation with shortened drug shelf life, and one of the major root causes of PS degradation is the host cell protein (HCP) derived lipase/esterase, which belong to the serine hydrolase family. Typically, PS degradation can only be observed in drug products after a long time of storage if very low levels of host cell protein impurity with PS degradation activities are present. In this study, PS80 degradation was observed in a monoclonal antibody (mAb) within 18 h at 5 °C with a low level of HCP presented (<20 ppm) based on ELISA quantitation. This observation suggested that a trace amount of unknown host cell protein(s) with strong enzymatic activity on polysorbate degradation was present in this drug substance. The activity-based protein profiling (ABPP) method with the ActivX FP serine hydrolase probe was employed to identify host cell proteins that can hydrolyze PS. Two hydrolases, liver carboxylesterase B-1-like protein (CES-B1L, A0A061I7X9) and liver carboxylesterase 1-like protein (CES-1L, A0A061IFE2) were identified with high confidence using the ABPP approach for the first time in a mAb drug substance during early stage development. PS80 became stable in the drug substance sample after the two hydrolases were depleted using the immobilized ActivX FP probe, confirming these two hydrolases were responsible for the rapid PS80 degradation. In addition, the PS80 degradation pattern was found to be equivalent to that generated by their human analog, human liver carboxylesterase-1 (hCES-1) and rabbit liver esterase (rLES). Overall, these results suggest that CES-B1L and CES-1L are the primary cause of PS80 degradation in this mAb drug.

Rapid Polysorbate 80 Degradation by Liver Carboxylesterase in a Monoclonal Antibody Formulated Drug Substance at Early Stage Development.

Host cell proteins (HCPs) impurities are critical quality attributes that have the potential to negatively impact the quality and safety profile of a biopharmaceutical product. Since HCPs often are present at low levels, developing highly sensitive analytical method for their identification and quantitation is critical for process optimization and improvement to reduce them in the final drug product. While an enzyme-linked immunosorbent assay (ELISA) can capture and quantify overall HCP levels, liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool to monitor individual HCP levels during the purification process development. The massive dynamic range of protein species present in a therapeutic antibody is a major challenge for mass spectrometry-based methods to detect low-abundance HCP impurities. This study reports a powerful strategy to identify HCPs in antibody drug substance by applying ProteoMiner enrichment with optimized conditions followed by shotgun proteomic analysis. Using this strategy, we observed that the low abundance HCPs were enriched up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 0.05 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 500 HCPs were confidently identified, which tripled the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were enriched between 100 and 400-fold, highlighting that our method enriches and equalizes all proteins thus improving the sensitivity of HCP identification and quantification.

Improved host cell protein analysis in monoclonal antibody products through ProteoMiner.

Polysorbates (PS) are surfactants commonly added in biologics formulations that can protect proteins from denaturation and aggregation. However, decreases in polysorbate 20 (PS20) content have been observed in some monoclonal antibody formulations, causing the formation of visible and/or subvisible particles that ultimately compromise the quality and stability of the therapeutic protein products. It was determined that the particles are mainly composed of free fatty acid, suggesting enzymatic hydrolysis of PS is responsible for the degradation of PS. Enrichment of host cell proteins (HCPs) by immunoprecipitation followed by shotgun proteomics have been utilized to identify the HCPs that can hydrolyze PS20. One HCP, sialate O-acetylesterase (SIAE), demonstrated strong enzymatic activity for PS20 degradation even at low concentration (<5 ppm level). Incubation of recombinant SIAE with PS20 resulted in a unique degradation pattern where the hydrolysis of monoester with short fatty acid chain (C12, C14) was observed but not the monoester with long fatty acid chain (C16, C18) or higher-order esters. SIAE was detected and quantitated in several formulated mAbs, and the amount of SIAE was positively correlated to PS20 degradation in these mAbs during incubation. Additional experiments also showed that when SIAE was depleted, PS20 degradation was diminished, suggesting a causality between SIAE and PS20 degradation. The lipase activity of SIAE is specific to PS20, but not to PS 80 (PS80), which contains monoesters with long chain fatty acid (C18) and higher-order esters. The specific esterase activity of SIAE on PS20 suggests a possible solution of using PS80 over PS20 to eliminate surfactant degradation in mAb products.

Degradation of Polysorbate 20 by Sialate O-Acetylesterase in Monoclonal Antibody Formulations.

Polysorbates (PSs) are surfactants commonly added to therapeutic protein drug product formulations to protect proteins from denaturation and aggregation during storage, transportation, and delivery. However, enzymatic hydrolysis of PSs has been recognized as the primary route of PS degradation in monoclonal antibody formulations, resulting in the release of free fatty acids that drive undesired particulate formation. Here, we present a rapid lipase activity assay with optimized incubation conditions for accurate quantitation of free fatty acids without a fatty acid extraction step. This assay can detect low levels of PS degradation (0.000024% PS20 degradation) within 1 day with minimal sample preparation. The levels of released free fatty acids were found to strongly correlate with the degree of PS20 degradation. The case study described herein suggests that this approach can detect low levels of PS20 degradation caused by sub-ppm lipase levels within 1 day, compared with the duration of 14 days needed for PS degradation assays based on two-dimensional liquid chromatography-charge aerosol detection.

Monitoring polysorbate hydrolysis in therapeutic proteins using an ultrasensitive extraction-free fatty acid quantitation method.

Polysorbates (PS) are excipients used in the biotech industry to stabilize monoclonal antibody (mAb) protein products. However, PS in drug product formulations can be degraded during storage and lead to particle formation because of the limited solubility of the free fatty acids released through the enzymatic hydrolysis of PS-a process driven by residual host cell proteins, especially lipases, that are co-purified with the drugs. When multiple lipases are present, it is very difficult to know the cause for PS degradation. In this study, we aim to determine the cause of PS degradation from two lipases, lysosomal acid lipase (LAL) and lipoprotein lipase (LPL).

Identification of the specific causes of polysorbate 20 degradation in monoclonal antibody formulations containing multiple lipases.

Correction to: Identification of the Specific Causes of Polysorbate 20 Degradation in Monoclonal Antibody Formulations Containing Multiple Lipases.

Polysorbates (PSs), including PS20 and PS80, are non-ionic surfactants widely used in the pharmaceutical industry to enhance drug solubility and stability. Despite their wide application, PSs are prone to degradation by either hydrolysis or oxidation in drug formulations during storage; therefore, a PS characterization method assessing protein products is needed for stability testing and for understanding the degradation pathway. In this article, we detail our protocol for sample preparation for forced degradation study and our instrumentation setup for PS profiling and quantitation in protein samples. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for forced degradation of polysorbate in protein samples Basic Protocol 2: Two-dimensional liquid chromatography coupled with charged aerosol detector or mass spectrometry to analyze polysorbate degradation.

The Characterization of Polysorbate.

Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination.

Analysis of Host Cell Proteins in Monoclonal Antibody Therapeutics Through Size Exclusion Chromatography.

Host cell proteins (HCPs) are process-related impurities that remain in therapeutic protein -at trace levels. HCPs must be closely monitored because they may be detrimental to drug product quality. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for detecting individual HCPs; however, HCP-derived peptides can be four to ten orders of magnitude less abundant than therapeutic protein-derived peptides in drug products, thus posing a major challenge in LC-MS detection. We previously demonstrated that low abundant HCPs can be enriched several hundreds fold through ProteoMiner. This study further improved the degree of enrichment by coupling limited digestion to ProteoMiner technology (PMLD). HCPs with low abundance were enriched 7694-fold, thus enabling detection of HCPs at concentrations as low as 0.002 ppm. A total of 850 HCPs were detected with high confidence from a NIST monoclonal antibody preparation, a number 40% higher than previously reported.

Ultrasensitive method for profiling host cell proteins by coupling limited digestion to ProteoMiner technology.

Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.

Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics.

Development of novel bispecific antibody (bsAb) platforms offers unprecedented opportunities for a wide variety of therapeutic applications. However, the expression and manufacturing of bsAbs with desired structures can be challenging. Owing to the uniqueness of each bsAb platform, more comprehensive and customized structural characterization is particularly important to understand the chemical or biological reactivity of bsAbs, as well as to guide process development, risk assessment, and manufacturing. In this work, we performed higher order structure characterization of the Regeneron bsAb platform with Fc site-specific substitutions through hydrogen deuterium exchange mass spectrometry (HDX-MS). Structural deprotection was identified at the C2-C3 interface in the Fc domain, owing to the site-specific substitutions. The structural deprotection was found to correlate with the decreased conformational stability of Fc domain. Under oxidative and thermal stress conditions, the Met residues located near the structurally deprotected region were identified to be susceptible to oxidation. In addition, the introduction of substitutions in the bsAb Fc resulted in a slight reduction of its binding affinity to the neonatal Fc receptor (FcRn). The detailed structural elucidation by HDX-MS improves understanding of the structure-property relationship of the Regeneron bsAb format, thus greatly aiding in process development, risk assessment, and manufacturing.

Article	Year
Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment. Analytical chemistry\| PubMed ID: 31999105	2020
Putative Phospholipase B-Like 2 is Not Responsible for Polysorbate Degradation in Monoclonal Antibody Drug Products. Journal of pharmaceutical sciences\| PubMed ID: 32534029	2020
Rapid Polysorbate 80 Degradation by Liver Carboxylesterase in a Monoclonal Antibody Formulated Drug Substance at Early Stage Development. Journal of pharmaceutical sciences\| PubMed ID: 32721471	2020
Improved host cell protein analysis in monoclonal antibody products through ProteoMiner. Analytical biochemistry\| PubMed ID: 32979367	2020
Degradation of Polysorbate 20 by Sialate O-Acetylesterase in Monoclonal Antibody Formulations. Journal of pharmaceutical sciences\| PubMed ID: 34487744	2021
Monitoring polysorbate hydrolysis in therapeutic proteins using an ultrasensitive extraction-free fatty acid quantitation method. Analytical biochemistry\| PubMed ID: 34801481	2022
Identification of the specific causes of polysorbate 20 degradation in monoclonal antibody formulations containing multiple lipases. Pharmaceutical research\| PubMed ID: 34981317	2022
Correction to: Identification of the Specific Causes of Polysorbate 20 Degradation in Monoclonal Antibody Formulations Containing Multiple Lipases. Pharmaceutical research\| PubMed ID: 35112230	2022
The Characterization of Polysorbate. Current protocols\| PubMed ID: 35857882	2022
Analysis of Host Cell Proteins in Monoclonal Antibody Therapeutics Through Size Exclusion Chromatography. Pharmaceutical research\| PubMed ID: 36071355	2022
Ultrasensitive method for profiling host cell proteins by coupling limited digestion to ProteoMiner technology. Analytical biochemistry\| PubMed ID: 36130653	2022
Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics. Analytical chemistry\| PubMed ID: 36977129	2023
Understanding the structure-property relationship of bispecific monoclonal antibodies with Fc site-specific substitutions. mAbs\| PubMed ID: 37357447	2023

Hui Xiao

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