Implementation of a High-Resolution Liquid Chromatography–Mass Spectrometry Method in Quality Control Laboratories for Release and Stability Testing of a Commercial Antibody Product

Sokolowska, Izabela; Mo, Jingjie; Pirkolachahi, Fatie Rahimi; McVean, Carol; Meijer, L.; Switzar, Linda; Balog, Crina I. A.; Lewis, Michael; Hu, Ping

doi:10.1021/acs.analchem.9b05036

Cited by 39 publications

(35 citation statements)

References 25 publications

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“…Middle-up LC-MS analysis holds significant potential to be used as a multi-attribute monitoring method (MAM) that could complement laborious released glycan approaches and complex MAM peptide-mapping workflows [26,27]. Despite the potential advantages, middle-up HILIC-MS analysis for N-glycan quantification has not yet been widely explored.…”

Section: Introductionmentioning

confidence: 99%

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

et al. 2021

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The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

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Section: Introductionmentioning

confidence: 99%

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

et al. 2021

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“…Mass spectrometry (MS) is now recognized as a major tool in protein structural characterization [5] and requested for example in ICH Q6B guideline (https://www.ich.org/page/search-index-ich-guidelines). Recent advances in high resolution MS instrumentation and have even led to the use of MS-based methods for quality control testing in a current Good Manufacturing Practice environment [6,7]. The continuous evolution of separative techniques and MS hyphenation enhances protein mixtures understanding and is widely applicable in mAbs assessments [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

Giorgetti

Beck

Leize‐Wagner

et al. 2020

Journal of Pharmaceutical and Biomedical Analysis

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Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 minutes and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)'2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)'2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.

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“…Multi-attribute methods (MAM), based on proteolytic digestion and subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of proteolytic peptides, are used to quantify a variety of quality attributes in protein therapeutics. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] These methods take advantage of the resolving power of a mass spectrometric (MS) detector and use the MS response of each peptide isoform for quantitation. Because of the specificity of these methods toward each clinically relevant quality attribute, they have gained extensive attention in the biopharmaceutical industry.…”

Section: Introductionmentioning

confidence: 99%

“…MS-based MAM has been performed on high-resolution mass spectrometers, [1][2][3][4][5][6][7][8][9][10][11] although some work has demonstrated the success of MAM on a low-resolution instrument. 12 Highresolution instruments, because of their high mass resolving power, are capable of resolving the analytes of interest from most interferences, and thereby providing superb analyte specificity.…”

Section: Introductionmentioning

confidence: 99%

An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics

Zhang

Chan

Richardson

et al. 2020

mAbs

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Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.

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Implementation of a High-Resolution Liquid Chromatography–Mass Spectrometry Method in Quality Control Laboratories for Release and Stability Testing of a Commercial Antibody Product

Cited by 39 publications

References 25 publications

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics

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