Dataset Information

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS.

ABSTRACT: Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies (mAbs) remains a challenge. Since residual HCPs might affect product stability or safety, constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg. The current standard analytical approach for this procedure is based on ELISA; however, this approach only measures the overall HCP content. Therefore, the use of orthogonal methods, such as liquid chromatography-mass spectrometry (LC-MS), has been established, as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present. In the present study, a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation, in combination with a data-independent acquisition (DIA) LC-MS analysis, was established. Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows, avoiding the need for dedicated instrumentation or personnel. Thereby, quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

SUBMITTER: Strasser L

PROVIDER: S-EPMC8740166 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS.

Strasser Lisa L Oliviero Giorgio G Jakes Craig C Zaborowska Izabela I Floris Patrick P Ribeiro da Silva Meire M Füssl Florian F Carillo Sara S Bones Jonathan J

Journal of pharmaceutical analysis 20210520 6

Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies (mAbs) remains a challenge. Since residual HCPs might affect product stability or safety, constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg. The current standard analytical approach for this procedure is based on ELISA; however, this approach only measures the overall HCP content. Therefore, the use ...[more]

PMID: 35028177

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Project description:BackgroundTherapeutic drug monitoring (TDM) systems generally use either liquid chromatography/tandem mass spectrometry (LC-MS/MS) or immunoassay, though both methodologies have disadvantages. In this study, we aimed to evaluate whether a CLAM-LC-MS/MS system, which consists of a sample preparation module directly connected to LC-MS/MS, could be used for clinical TDM work for immunosuppressive drugs in whole blood, which requires a hemolytic process. For this purpose, we prospectively validated this system for clinical measurement of tacrolimus and cyclosporin A in patients' whole blood. The results were also compared with those of commercial immunoassays.MethodsWhole blood from patients treated with tacrolimus or cyclosporin A at the Department of Nephrology and Departments of Rheumatology, Kanazawa University Hospital, from May 2018 to July 2019 was collected with informed consent, and drug concentrations were measured by CLAM-LC-MS/MS and by chemiluminescence immunoassay (CLIA) for tacrolimus and affinity column-mediated immunoassay (ACMIA) for cyclosporin A. Correlations between the CLAM-LC-MS/MS and immunoassay results were analyzed.ResultsTwo hundred and twenty-four blood samples from 80 patients were used for tacrolimus measurement, and 76 samples from 21 patients were used for cyclosporin A. Intra- and inter-assay precision values of quality controls were less than 7%. There were significant correlations between CLAM-LC-MS/MS and the immunoassays for tacrolimus and cyclosporin A (Spearman rank correlation coefficients: 0.861, 0.941, P < 0.00001 in each case). The drug concentrations measured by CLAM-LC-MS/MS were about 20% lower than those obtained using the immunoassays. CLAM-LC-MS/MS maintenance requirements did not interfere with clinical operations. Compared to manual pretreatment, automated pretreatment by CLAM showed lower inter-assay precision values and greatly reduced the pretreatment time.ConclusionsThe results obtained by CLAM-LC-MS/MS were highly correlated with those of commercial immunoassay methods. CLAM-LC-MS/MS offers advantages in clinical TDM practice, including simple, automatic pretreatment, low maintenance requirement, and avoidance of interference.

Project description:Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Dataset Information

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS.

Publications

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS.

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