Identification Of Host Cell Proteins Research Articles

Analytical Tools for HCP Detection, Identification, and Quantitation.

Identifying and quantifying host cell proteins (HCPs) are crucial in developing and manufacturing biopharmaceutical products. ELISA has been used widely for identifying and quantifying HCPs in biopharmaceutical products. Liquid chromatography-mass spectrometry (LC-MS) has recently emerged as an orthogonal tool for HCP analysis, providing comprehensive and quantitative information on individual HCPs. This chapter explores the advancements in LC-MS/MS methodologies for HCP analysis, including sample preparation, data acquisition modes, and data analysis strategies. It also discusses the challenges of HCP identification and quantitation, and recent innovations to overcome these limitations. The application of LC-MS/MS methods in HCP profiling holds significant promise for improving process control and ensuring product safety in biopharmaceutical manufacturing.

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1–2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.

Characterization of Recombinantly-Expressed Hydrolytic Enzymes from Chinese Hamster Ovary Cells: Identification of Host Cell Proteins that Degrade Polysorbate

Enzymatic hydrolysis of polysorbate in drug products is a major challenge for the biopharmaceutical industry. Polysorbate hydrolysis caused by host cell proteins (HCPs) co-purified during bioprocessing can reduce the protective effects of the surfactant for the active pharmaceutical ingredient and cause the accumulation of low-solubility degradation products over the long-term storage. The identities of such HCPs are elusive due to their extremely low concentrations after the efficient purification processes of most biopharmaceuticals. In this work, 20 enzymes—selected for their known or putative hydrolytic activity and potential to degrade polysorbate—were recombinantly expressed, purified, and characterized via orthogonal methods. First, these recombinant HCPs were assessed for hydrolytic activity against a fluorogenic esterase substrate in a recently-developed, high-throughput assay. Second, these HCPs were screened for hydrolytic activity against polysorbate in a representative mAb formulation. Third, HCPs that displayed hydrolytic activities in the first two assays were subjected to more detailed characterization of their enzyme kinetics against polysorbates. Finally, these HCPs were evaluated for substrate specificity towards different sub-species of polysorbates. This work provides critical new insights for targeted LC-MS/MS approaches for identification of relevant polysorbate-degrading enzymes and supports improvements to remove such HCPs, including knockouts or targeted removal during purification.

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

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. We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs. Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions. This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Identification of host cell proteins that interact with the M protein of Pigeon paramyxovirus type 1

Pigeon paramyxovirus type 1 (PPMV-1) belongs to the avian paramyxovirus type 1 group of viruses, which can cause tremors, torticollis, and respiratory signs in domestic and wild pigeons. The M protein of PPMV-1 is a multifunctional structural protein. It not only helps in the assembly, budding, and positioning of the virus but also inhibits the host's immune response and promotes replication of the virus in the host. In this study, the GST pull-down method was used to screen host proteins that interact with PPMV-1 M protein, and then mass spectrometry (MS) was used to analyse the screened host proteins. Enrichment analysis of the differentially expressed genes showed that the 77 screened proteins were highly associated with the gene ontology categories: protein synthesis, metabolism, and cell signalling pathway transduction. We selected NIMA-related kinase 7 (NEK7) as the candidate protein for co-localization analysis and co-immunoprecipitation verification. The results revealed that PPMV-1 M protein interacts with NEK7 of the host cell. This interactome study of PPMV-1 M protein will serve to clarify its function during viral replication and will provide a crucial theoretical basis for studying the pathogenic mechanism of PPMV-1.

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

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.

Toward unbiased identification and comparative quantification of host cell protein impurities by automated iterative LC–MS/MS (HCP-AIMS) for therapeutic protein development

Host cell proteins (HCPs) are process-related impurities expressed by the host cells used for production of therapeutic proteins. Although an extensive purification process removes most of the HCPs, residual HCPs are commonly present in protein therapeutics. If not well-controlled, certain HCPs may be present in the product, impacting drug stability, and potentially affecting product safety leading to safety risks for patients. Therefore, as a critical quality attribute, the levels of HCPs must be closely monitored during drug development and determined in the final drug substance at release. Liquid chromatography-mass spectrometry (LC–MS) as an orthogonal approach to traditional ELISA for HCP analysis has shown tremendous value in drug process development and analytical characterization by providing additional critical information, such as protein identity and relative abundance of individual HCPs. To meet the challenges in HCP analysis during drug development, especially downstream process development, which entails fast turnaround time and robustness while identifying high level of HCPs and their clearance trend for further purification development, we have developed HCP-automated iterative MS (HCP-AIMS): it is a simple, automated and robust HCP analysis workflow with deep and unbiased identification and relative quantification capability. This HCP-AIMS approach only requires easy direct digestion of the samples without enrichment or pre- treatment. With the fully automated precursor ion exclusion in MS/MS mode, low abundance HCP peptides could be selected for MS/MS analysis in iterative replicates, and therefore, the identification of HCPs at low abundance can be achieved. Using an in-house mAb with various levels of spiked-in HCPs as well as the NIST mAb, we were able to achieve unbiased identification and quantitation of HCPs as low as 10 ppm level. Furthermore, robustness of the HCP-AIMS approach was also confirmed for the feasibility of large-scale and high-throughput analysis.

Improved host cell protein analysis in monoclonal antibody products through ProteoMiner

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.

Fast, Robust, and Sensitive Identification of Residual Host Cell Proteins in Recombinant Monoclonal Antibodies Using Sodium Deoxycholate Assisted Digestion.

Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7-8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development.Graphical abstract

Identification of host cell proteins that interact with the M protein of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes severe diarrhea in pigs of all ages and a high fatality rate in neonates. The PEDV membrane protein (M) plays crucial roles in viral assembly, viral budding and host immune regulation, most likely by interacting with host cell proteins that have yet to be identified. In this study, co-immunoprecipitation (Co-IP) using an M-specific monoclonal antibody, coupled with LC-MS/MS, was employed to identify M protein-interacting proteins in PEDV-infected cells. Three viral proteins (S, E and ORF3) and 218 host cell proteins were identified as putative M-interacting partners. Bioinformatic analysis showed that the identified host cell proteins were related to 131 signal pathways and 10 biological processes. In addition, interaction between translation initiation factor 3(eIF3L) and M protein was validated by Co-IP. Down-regulation of eIF3L expression significantly increased viral production, which suggests that eIF3L could be a negative regulator in PEDV replication. This interactome study of the PEDV M protein will serve to clarify its function during viral replication.

Improved identification of host cell proteins in a protein biopharmaceutical by LC–MS/MS using the ProteoMiner™ Enrichment Kit

Host cell proteins (HCPs) in biotherapeutics can be identified by the use of enzymatic digestion and LC–MS/MS analysis. However, the major challenge is that HCPs are often present at very low levels in relation to the protein drug (low ppm-levels). In this study, the ProteoMiner™ Enrichment Kit (Bio-Rad) was evaluated as a strategy to enable identification of HCPs by LC–MS/MS by enrichment of low-abundant HCPs and a simultaneous depletion of the high-abundant product protein.A recombinant protein produced in Chinese hamster ovary (CHO) cells was spiked with six standard proteins at varying concentrations (10−1000 ppm). The sample was split into two aliquots; one that was prepared with the ProteoMiner™ Enrichment Kit and one control, where the enrichment procedure was omitted. The ProteoMiner™ Enrichment Kit was combined with the ProteoMiner Sequential Elution Large-Capacity Kit (Bio-Rad), eluting the proteins into four fractions. The samples were then digested with trypsin and analyzed with LC–MS/MS. In addition, multiple reaction monitoring (MRM) was applied to obtain an estimate of the protein abundance of HCPs and spiked proteins. The results demonstrated that with the untargeted LC–MS/MS method, 30 HCPs and four of the six spiked standard proteins were identified in the four fractions. The spiked standard proteins were identified down to 30 ppm in the ProteoMiner treated samples, while no HCPs and only the most abundant standard protein (≈1000 ppm) were identified in the non-enriched control sample. MRM assays were developed for 14 out of the 30 identified HCPs. All targeted HCPs and five of the six standard proteins were detected in all fractions as well as in the control sample by MRM. There was an acceptable agreement between estimated concentrations of spiked standard proteins and expected values. An 80–700 fold enrichment of individual HCPs was observed in the fractions. In conclusion, the results clearly demonstrated that the ProteoMiner technology can be used for enriching HCPs in biotherapeutics, enabling their identification by LC–MS/MS.

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

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.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast.

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy. Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation. Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies.

Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross-linking, followed by incubation with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP–mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme-linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1114–1124, 2014

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein.