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Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

ABSTRACT: Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.

SUBMITTER: Bomans K

PROVIDER: S-EPMC3842259 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

Bomans Katrin K Lang Antje A Roedl Veronika V Adolf Lisa L Kyriosoglou Kyrillos K Diepold Katharina K Eberl Gabriele G Mølhøj Michael M Strauss Ulrike U Schmalz Christian C Vogel Rudolf R Reusch Dietmar D Wegele Harald H Wiedmann Michael M Bulau Patrick P

PloS one 20131127 11

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host ...[more]

PMID: 24312330

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Project description:Numerous reverse transcription polymerase chain reaction (RT-PCR) tests have emerged over the past year as the gold standard for detecting millions of cases of SARS-CoV-2 reported daily worldwide. However, problems with critical shortages of key reagents such as PCR primers and RNA extraction kits and unpredictable test reliability related to high viral replication cycles have triggered the need for alternative methodologies to PCR to detect specific COVID-19 proteins. Several authors have developed methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to confirm the potential of the technique to detect two major proteins, the spike and the nucleoprotein, of COVID-19. In the present work, an S-Trap mini spin column digestion protocol was used for sample preparation prodromal to LC-MS/MS analysis in multiple reactions monitoring ion mode (MRM) to obtain a comprehensive method capable of detecting different viral proteins. The developed method was applied to n. 81 oro/nasopharyngeal swabs submitted in parallel to quantitative reverse transcription PCR (RT-qPCR) assays to detect RdRP, the S and N genes specific for COVID-19, and the E gene for all Sarbecoviruses, including SARS-CoV-2 (with cycle negativity threshold set to 40). A total of 23 peptides representative of the six specific viral proteins were detected in the monitoring of 128 transitions found to have good ionic currents extracted in clinical samples that reacted differently to the PCR assay. The best instrumental response came from the FLPFQFGR sequence of spike [558-566] peptide used to test the analytical performance of the method that has good sensitivity with a low false-negative rate. Transition monitoring using a targeted MS approach has the great potential to detect the fragmentation reactions of any peptide molecularly defined by a specific amino acid sequence, offering the extensibility of the approach to any viral sequence including derived variants and thus providing insights into the development of new types of clinical diagnostics.

Project description:Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

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Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

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Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

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