Quantitative Proteomic Analysis of Residual Host Cell Protein Retention Across Adeno- Associated Virus (AAV) Affinity Chromatography
Ontology highlight
ABSTRACT:
ORGANISM(S): Cellular Organisms
SUBMITTER: Kelvin H. Lee
PROVIDER: PXD056294 | JPOST Repository | Thu Dec 12 00:00:00 GMT 2024
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| AAV2_B1.zip | Other | |||
| AAV2_B2.zip | Other | |||
| AAV5_B1.zip | Other | |||
| AAV5_B2.zip | Other | |||
| AAV8_B1.zip | Other |
Items per page: 5 1 - 5 of 13 |
Shared Molecules
Only show the datasets with similarity scores above: 0.5
Threshold
0.5
Similar Datasets
Project description:Analysis of residual host cell proteins (HCPs) in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. To address these challenges, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) data was acquired on four AAV serotypes (2, 5, 8, and 9) produced with HEK293 cells and purified using POROS™ CaptureSelect™ AAVX affinity chromatography with two different analysis software (Skyline and DIA-NN) and instruments (Sciex TripleTOF 6600 and ZenoTOF 7600). An improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods well-suited for rAAV HCP analytics was developed by comparing number of protein and precursor identification, HCP quantification and quantification coefficient of variance (CV).
2025-04-23 | PXD060891 | JPOST Repository
Project description:Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.
2020-03-19 | GSE144624 | GEO
Project description:There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels.1-6 In some cases, regulatory agencies require the identification as well as the quantification of HCPs in drug products for risk assessment, and this is an active and growing topic of conversation in industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in the significantly shorter turnaround time than previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Due to its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody (mAb) reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances (DS) and drug products (DP).
2020-06-02 | PXD018427 | Pride
Project description:Monitoring of host cell proteins (HCPs) during the manufacture of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug product. ELISA assays are still the current gold standard for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) has emerged as an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography (LC)-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. In this study, we developed a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performance of the HCP Profiler solution was compared to more conventional standard protein spikes and the DIA approach was benchmarked against classical data-dependent acquisition (DDA) using samples collected at various stages of the manufacturing process. Finally, we further explored the possibility to use spectral library-free DIA. As a result, our method showed accurate and reproducible (coefficients of variation (CVs) < 10%) quantification of HCPs while reaching a sensitivity down to the sub-ng/mg mAb level.
2023-07-20 | PXD029305 | Pride
Project description: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 may often arebe present at low levels, developing therefore highly a sensitive analytical method is needed to for their identification and quantitation is critical for process optimization and improvement to reduce them in the final drug product.ensure the safety and efficacy of the biopharmaceutical 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 an antibody drug substance by applying ProteoMiner enrichment 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 to 400-fold, highlighting that our method enriches and equalizes all proteins thus improving the sensitivity of HCP identification and quantification.
2021-06-02 | PXD019497 | JPOST Repository
Project description: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 cut-off (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 by 1,000-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 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.
2020-11-08 | PXD016194 | JPOST Repository
Project description:In this study, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation prior to data dependent acquisition (DDA) and gas phase fractionation (GPF) prior to data independent acquisition (DIA) LC-MS/MS analysis. Native digestion followed by LC-MS/MS with FAIMS allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been applied to commercial DPs and demonstrate their ability to dig deeper into the HCP landscape with the identification of 60 and 67 HCPs, and accurate quantification of 29 and 31 of these impurities in nivolumab and trastuzumab respectively, with sensitivity down to the sub-ng/mg of mAb level
2023-05-07 | PXD039585 | Pride
Project description:In this study, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation prior to data dependent acquisition (DDA) and gas phase fractionation (GPF) prior to data independent acquisition (DIA) LC-MS/MS analysis. Native digestion followed by LC-MS/MS with FAIMS allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been applied to commercial DPs and demonstrate their ability to dig deeper into the HCP landscape with the identification of 60 and 67 HCPs, and accurate quantification of 29 and 31 of these impurities in nivolumab and trastuzumab respectively, with sensitivity down to the sub-ng/mg of mAb level.
2023-05-07 | PXD039582 | Pride
Project description:In this study, we developed powerful and reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, library-free DIA acquisition method and stringent validation criteria. The performances of several preparation protocols and DIA versus classical DDA were evaluated using a series of four commercially available Drug Products. Depending on the selected protocols, the user has access to different information: on the one hand, a deep profiling of tens of identified HCPs, and on the other hand an accurate and reproducible (CV<12%) quantification of major HCPs. Overall, a final global HCP amount of a few tens of ppm in these mAb samples was measured, while reaching a sensitivity down to the sub-ppm level. Thus, this straightforward and robust approach can be intended as a routine quality control for whichever Drug Products’ analysis.
2020-10-19 | PXD019668 | Pride
Project description:Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enablesenables highly sensitive detection of HCPs, but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 minutes, but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low LOD and LOQ (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in biopharmaceuticals.
2025-05-02 | PXD053135 | JPOST Repository