Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples
The success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2020-01-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2019.1702263 |
| _version_ | 1818023781517492224 |
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| author | Zachary D. Dunn Jayesh Desai Gabriel M. Leme Dwight R. Stoll Douglas D. Richardson |
| author_facet | Zachary D. Dunn Jayesh Desai Gabriel M. Leme Dwight R. Stoll Douglas D. Richardson |
| author_sort | Zachary D. Dunn |
| collection | DOAJ |
| description | The success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical quality attributes (CQAs) during production. The current workflow to measure CQAs of antibodies involves multiple one-dimensional liquid chromatography methods, including Protein-A (ProA), ion-exchange (IEX), reversed-phase, size exclusion (SEC), hydrophilic interaction, and hydrophobic interaction (HIC). Recent advances in commercial two-dimensional liquid chromatography (2D-LC) affords an opportunity to perform two separations at once to measure multiple CQAs in a single assay. Here, we describe the development of a 2D ProA–SEC method using entirely commercially available instrumentation. Each individual separation and the transfer of material between dimensions were optimized to develop a method that measures titer and aggregation of a target antibody from harvested cell culture fluid in under 5 min. We determined the effects of each parameter of the method on mAb recovery and stability, as well as speed, robustness, resolution, and accuracy of the aggregate amount detected in the second dimension (2D). While there are still sources of error caused by hardware limitations, our rapid ProA-SEC method is an effective screening tool with a significant throughput advantage over previously described methods. Additionally, this work serves as a basis for developing other 2D-LC methods with ProA as the first dimension (1D) separation coupled with different 2D separation, such as ProA-IEX and ProA-HIC. |
| first_indexed | 2024-12-10T03:49:47Z |
| format | Article |
| id | doaj.art-06b9363b187149d795c4f4cf0af26491 |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-12-10T03:49:47Z |
| publishDate | 2020-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-06b9363b187149d795c4f4cf0af264912022-12-22T02:03:18ZengTaylor & Francis GroupmAbs1942-08621942-08702020-01-0112110.1080/19420862.2019.1702263Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samplesZachary D. Dunn0Jayesh Desai1Gabriel M. Leme2Dwight R. Stoll3Douglas D. Richardson4Analytical Research and Development, Merck & Co. Inc., Kenilworth, NJ, USAProcess Research and Development, Merck & Co. Inc., Kenilworth, NJ, USADepartment of Chemistry, Gustavus Adolphus College, St. Peter, MN, USADepartment of Chemistry, Gustavus Adolphus College, St. Peter, MN, USAAnalytical Research and Development, Merck & Co. Inc., Kenilworth, NJ, USAThe success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical quality attributes (CQAs) during production. The current workflow to measure CQAs of antibodies involves multiple one-dimensional liquid chromatography methods, including Protein-A (ProA), ion-exchange (IEX), reversed-phase, size exclusion (SEC), hydrophilic interaction, and hydrophobic interaction (HIC). Recent advances in commercial two-dimensional liquid chromatography (2D-LC) affords an opportunity to perform two separations at once to measure multiple CQAs in a single assay. Here, we describe the development of a 2D ProA–SEC method using entirely commercially available instrumentation. Each individual separation and the transfer of material between dimensions were optimized to develop a method that measures titer and aggregation of a target antibody from harvested cell culture fluid in under 5 min. We determined the effects of each parameter of the method on mAb recovery and stability, as well as speed, robustness, resolution, and accuracy of the aggregate amount detected in the second dimension (2D). While there are still sources of error caused by hardware limitations, our rapid ProA-SEC method is an effective screening tool with a significant throughput advantage over previously described methods. Additionally, this work serves as a basis for developing other 2D-LC methods with ProA as the first dimension (1D) separation coupled with different 2D separation, such as ProA-IEX and ProA-HIC.https://www.tandfonline.com/doi/10.1080/19420862.2019.1702263Two-dimensional liquid chromatographyprotein-Asize exclusion chromatographyaggregationmonoclonal antibodytiter |
| spellingShingle | Zachary D. Dunn Jayesh Desai Gabriel M. Leme Dwight R. Stoll Douglas D. Richardson Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples mAbs Two-dimensional liquid chromatography protein-A size exclusion chromatography aggregation monoclonal antibody titer |
| title | Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| title_full | Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| title_fullStr | Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| title_full_unstemmed | Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| title_short | Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| title_sort | rapid two dimensional protein a size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples |
| topic | Two-dimensional liquid chromatography protein-A size exclusion chromatography aggregation monoclonal antibody titer |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2019.1702263 |
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