Multi-Attribute Method for Quality Control of Therapeutic Proteins

Rogstad, Sarah; Yan, Haoheng; Wang, Xiaoshi; Powers, David N.; Brorson, Kurt; Damdinsuren, Bazarragchaa; Lee, Sau

doi:10.1021/acs.analchem.9b03808

Cited by 93 publications

(124 citation statements)

References 15 publications

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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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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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“…antibody screening | high throughput | intact protein mass spectrometry | electrospray U nambiguous characterization of analytes from complex matrices with high content information in a label-free format continues to expand the application of mass spectrometry (MS) in drug discovery (1)(2)(3). With the need for high accuracy, sensitivity, and selectivity, the rapidly improving MS instrumentations are emerging at the forefront of analyzing analytes with limited alternative assays for biologics developability (4)(5)(6), biotransformation, and high-throughput screening (HTS).…”

mentioning

confidence: 99%

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Sawyer¹,

Srikumar²,

Carver³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFiretime-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.antibody screening | high throughput | intact protein mass spectrometry | electrospray U nambiguous characterization of analytes from complex matrices with high content information in a label-free format continues to expand the application of mass spectrometry (MS) in drug discovery (1-3). With the need for high accuracy, sensitivity, and selectivity, the rapidly improving MS instrumentations are emerging at the forefront of analyzing analytes with limited alternative assays for biologics developability (4-6), biotransformation, and high-throughput screening (HTS). For ultra-high-throughput screening, matrix-assisted laser desorption/ionization (MALDI) is amenable to speeds >100,000 samples per day (7-11). Through incorporating self-assembled monolayers for MALDI-time-of-flight (TOF) (SAMDI) technology (12-14), it is also possible to infer small molecule noncovalent hit identification from MALDI-MS detection under native conditions. While such MALDI-MS approaches have the capability for screening small molecule and peptide analytes, larger molecular weight proteins suffer from limited mass resolution and quantitative challenges. Alternatively, electrospray ionization MS (ESI-MS) can provide isotopic resolution for molecules as large as antibodies (15). Although modern mass spectrometers can scan as rapidly as tens of microseconds, highthroughput antibody analyses using ESI-MS is challenged by the relatively low ion sampling rate from chromatographic elution coupled to MS. For rapid sampling, Agilent RapidFire MS (RF-MS) utilizes a rapid trap and elute strategy to enable desalting and ion sampling coupled to a MS ion source. RF-MS has been shown to afford HTS triage for small molecules and proteins <30 kDa from biochemical buffers as fast as 15 s as opposed to minutes observed with conventional chromatography (16). Altern...

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“…The U.S. FDA recently reported that nearly all protein therapeutic biologics license applications approved between 2000 and 2015 used mass spectrometry for the characterization of drug substance and impurities (Rogstad et al, 2017). Liquid chromatography‐mass spectrometry (LC‐MS) based multiattribute methods for simultaneous monitoring of the glycan profile, charge variants, and purity of biotherapeutics was first reported by Rogers et al (2015), and has since gained significant utility in bioprocess development (Rogstad et al, 2019). As multiattribute LC‐MS methods can measure multiple CQAs in a single run, it eliminates the need for typical chromatographic tests such as hydrophilic interaction liquid chromatography for glycan profiling, cation exchange chromatography for charge variant analysis, and reduced capillary electrophoresis for purity analysis.…”

Section: Process Analytical Technologiesmentioning

confidence: 99%

“…Due to the technological maturation of on‐column trypsin digestion technologies and advanced sampling techniques such as the μSI System® from FIAlab®, it is now feasible to monitor product quality attributes such as glycans and other posttranslational modifications in an on‐line fashion (Toth, Kuklenyik, Morris, & Barr, 2019). Despite the aforementioned attractive features of LC‐MS based multi‐CQA monitoring tools, there are unique challenges associated with these systems, including the complexity of the technology, complicated method life cycle management, and method validation complications with new peak detection (Rogstad et al, 2019).…”

Section: Process Analytical Technologiesmentioning

confidence: 99%

Technology outlook for real‐time quality attribute and process parameter monitoring in biopharmaceutical development—A review

Wasalathanthri

Rehmann

Song

et al. 2020

Biotech & Bioengineering

114

101

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Real‐time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real‐time release (RTR) of biopharmaceuticals. A well‐defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real‐time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated‐data piping, analysis, aggregation, visualization, and smart utility of data for advanced‐data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated‐sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced‐analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

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Multi-Attribute Method for Quality Control of Therapeutic Proteins

Cited by 93 publications

References 15 publications

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

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

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Technology outlook for real‐time quality attribute and process parameter monitoring in biopharmaceutical development—A review

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