Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay
A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that...
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2024.2379903 |
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| author | Linlin Dong Susan Chen Konstantin Piatkov Dong Wei Mark G. Qian |
| author_facet | Linlin Dong Susan Chen Konstantin Piatkov Dong Wei Mark G. Qian |
| author_sort | Linlin Dong |
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| description | A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics. |
| format | Article |
| id | doaj-art-f29ac644e16c4755a15767df2a17402a |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
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| series | mAbs |
| spelling | doaj-art-f29ac644e16c4755a15767df2a17402a2025-01-31T04:19:38ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2024.2379903Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assayLinlin Dong0Susan Chen1Konstantin Piatkov2Dong Wei3Mark G. Qian4Department of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USADepartment of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USADepartment of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USADepartment of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USADepartment of Drug Metabolism, Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., Cambridge, MA, USAA sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.https://www.tandfonline.com/doi/10.1080/19420862.2024.2379903Immunoaffinity enrichmentLC/MS/MSmonoclonal antibodymouse plasmapepsin |
| spellingShingle | Linlin Dong Susan Chen Konstantin Piatkov Dong Wei Mark G. Qian Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay mAbs Immunoaffinity enrichment LC/MS/MS monoclonal antibody mouse plasma pepsin |
| title | Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay |
| title_full | Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay |
| title_fullStr | Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay |
| title_full_unstemmed | Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay |
| title_short | Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay |
| title_sort | quantifying laga mutated mouse igg2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity lc ms ms assay |
| topic | Immunoaffinity enrichment LC/MS/MS monoclonal antibody mouse plasma pepsin |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2024.2379903 |
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