Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

Guapo, Felipe; Strasser, Lisa; Millán-Martín, Silvia; Anderson, Ian; Bones, Jonathan

doi:10.1016/j.jpba.2021.114427

Cited by 23 publications

(24 citation statements)

References 21 publications

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“…PTMs on biotherapeutic proteins are monitored as potential CQAs to inform process development, batch consistency, product stability, and more, as their presence can influence product quality, efficacy, and potentially patient safety. ,− Comprehensive PTM analysis is crucial for in-depth characterization of full-length AAV VPs, as it aids in the proper identification of different VP proteoforms present and any potential consequences their presence might cause. For PTM determination, peptide mapping and relative PTM quantitation of AAV2 using on-bead pepsin digestion and nano-LC separation were performed in parallel to full-length VP analysis, following the procedure described by Guapo et al One hundred percent sequence coverage was obtained after filtering identified peptides in BPF as described in the section describing AAV peptide mapping data processing for improved peptide mapping confidence (Figure S1). Here, it should be noted the VP1 sequence searched for peptide mapping was A2-735 as it is well-known that for AAV2 the N-terminal methionine (M, Met) residue is cleaved within the cell ,,, (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

Smith,

Guapo,

Strasser

et al. 2023

J. Proteome Res.

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Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) have become a critical attribute monitored to evaluate product quality. Liquid chromatography−mass spectrometry (LC−MS) analysis of intact AAV VPs provides both quick and reliable serotype identification as well as proteoform information on each VP. Incorporating these analytical strategies into rapid good manufacturing practice (GMP)-compliant workflows containing robust, but simplified, data processing methods is necessary to ensure effective product quality control (QC) during production. Here, we present a GMP-compliant LC− MS workflow for the rapid identification and in-depth characterization of AAVs. Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic acid as a mobile phase modifier is utilized to achieve the intact separation and identification of AAV VPs and their potential proteoforms. Peptide mapping is performed to confirm PTMs identified during intact VP analysis and for indepth PTM characterization. The intact separations platform is then incorporated into a data processing workflow developed using GMP-compliant software capable of rapid AAV serotype identification and, if desired, specific serotype PTM monitoring and characterization. Such a platform provides product QC capabilities that are easily accessible in a regulatory setting.

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Section: Resultsmentioning

confidence: 99%

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

Smith,

Guapo,

Strasser

et al. 2023

J. Proteome Res.

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“…Interestingly, PTMs were observed on all three VP subunits of AAV8, but rather than oxidation, phosphorylation was observed. Characterization of PTMs is important because they could have implications to the overall functionality and stability of AAV formulations. , …”

Section: Results and Discussionmentioning

confidence: 99%

“…Characterization of PTMs is important because they could have implications to the overall functionality and stability of AAV formulations. 45,46 DTIMS Analysis. To confirm and further characterize the PTMs and VP amino acid sequences of the denatured VPs, IMS-MS was employed, following the LC separation.…”

Section: ■ Introductionmentioning

confidence: 99%

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods

Ryan,

Kostelic,

Hsieh

et al. 2023

J. Am. Soc. Mass Spectrom.

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“…It is also known that trypsin is not effective in digesting adeno-associated virus capsid proteins. 16 …”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control

Yang

Zhang

Buettner

et al. 2023

mAbs

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The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.

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Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

Cited by 23 publications

References 21 publications

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control

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