An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules

The challenge with accurate mass calculation for large protein molecules is the mass defect associated with the measurement. As a result, only average masses can be measured. Minor elemental mass differences may accumulate, causing significant discrepancies in large paratherapeutic molecules like monoclonal antibodies.

Our tool explicitly addresses the challenge in the consistent mass calculation for antibody-based therapeutics, such as mabs, bispecifics, and antibody drug conjugates. Researchers in the commercial biopharmaceutical development space have to rely on manual calculation or tools that don't depend on communication with external servers. Due to the involvement of proprietary information, existing tools either require a license, or are not built considering the specific needs of biopharmaceutical development.

Mabscale offers a user-friendly interface and adaptable open source framework for users and developers. It supports maintaining multiple versions of elemental masses and modification lists, and allows list sharing to maintain consistency in calculations. The code can be shared on an enterprise level using internal servers or cloud-based deployment, with controlled access to protect proprietary information.

To begin, open the software application by double clicking on the icon for an executable file. Enter the heavy chain and light chain sequences in the text boxes under the one column without any spaces. For bispecific antibodies, add additional heavy or light chains in the second set of text boxes under the two column.

Leave these boxes blank for monoclonal antibodies with identical heavy chains and light chains. Check the end terminal cyclization and C terminal clipping checkboxes if these heavy chain terminal variants are applicable. Add chemical modifications, including linker and payload for antibody drug conjugates to the heavy and light chain chemical modification boxes using elemental compositions such as calcium chloride.

These modifications will be added to the respective protein subunit or chain. Specify the number of disulfide bonds in the protein molecules in the text box marked, Total Number of Disulfides. Enter the number of unreduced heavy chain disulfide bonds into the unreduced heavy chain disulfides text box, and the number of unreduced light chain disulfide bonds into the unreduced light chain disulfides text box, depending on the extent of reduction.

If glycosylation is present on the monoclonal antibodies light chain, check The Light Chain is Glycosylated checkbox. Click on the Browse button to select an output folder for the output folder text box, and enter the output file name, without a file extension, into the Excel file, no extension text box. Then click on the Submit button.

A mass calculation report created message will appear on the screen and the output file can be found in the user designated folder. The app will close automatically. The chemical compositions, calculated masses, measured masses, and mass errors of various monoclonal antibodies are summarized in this table.

A commercially available monoclonal antibody standard was selected to represent a conventional monoclonal antibody with identical heavy chains, identical light chains, and one N-linked glycosylation site in the FC region. A monoclonal antibody with an additional light chain N-linked glycosylation, a bispecific monoclonal antibody, and an antibody drug conjugate monoclonal antibody were also chosen to widen the application usage. The small mass errors between the calculated masses and measured masses were within the normal mass error acceptance criteria, suggesting that the calculated masses were accurate.