Dataset Information

Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.

ABSTRACT: To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.

SUBMITTER: Iacob RE

PROVIDER: S-EPMC3970822 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.

Iacob Roxana E RE Bou-Assaf George M GM Makowski Lee L Engen John R JR Berkowitz Steven A SA Houde Damian D

Journal of pharmaceutical sciences 20131017 12

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry ...[more]

PMID: 24136070

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Project description:Patents of some therapeutic monoclonal antibodies (mAb) used for cancer treatment are ending soon, allowing for the entry of similar analogs (biosimilars) onto the market. To have a biosimilar approved by health agencies, the manufacturer must typically demonstrate functional and physicochemical similarity between the biosimilar and the approved reference product. Functional similarity is often validated with cell-based assays, but the information gained is limited. In contrast, RNA-seq methods enable a sensitive transcriptomic analysis, providing detailed information on pathways and cellular responses. Trastuzumab inhibits signaling of the cell-surface receptor HER2, which is overexpressed in approximately 30% of all breast cancer patients. Here, we compare the functional effect of the mAb Trastuzumab and a corresponding biosimilar. The BT-474 breast cancer cell line was treated with the reference product, Trastuzumab (Herceptin®), or a proposed biosimilar (ApoTras), and the cellular transcriptomes were analyzed by RNA-seq. Functional similarity was assessed using two statistical contrasts. One contrast evaluated the mechanism of action by comparing treated samples (Her and ApoTras, n = 19) vs. untreated controls (n = 10), which identified 2623 differentially expressed genes (DEG) at a minimum fold change of 1.25. The other contrast directly compared ApoTras (n = 9) vs. Her (n = 10) to determine differences in expression between treatments and identified 24 DEG using a 1.25 fold- change threshold. This low DEG number reveals a very high similarity of effect of both mAb treatments on the cancer cells. A gene set overrepresentation analysis of DEG for mAb treatments revealed mechanisms of action, which were consistent with known trastuzumab effects. Supporting the small number of changes between both treatments, a post-hoc power analysis for the between-treatment comparison estimated power of 0.88 to detect a gene expression fold-changes of 2.0. To summarize these data, we introduce an overall similarity index (SI) to quantify treatment similarity based on changes in gene expression. Using statistical contrasts with RNA-seq analysis provides a powerful tool for the comparison of biological effects of biosimilar and originator compounds and can be broadly used for functional comparisons of drug treatments.

Dataset Information

Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.

Publications

Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.

Similar Datasets

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