running the mabscale application to calculate the masses of monoclonal antibody-based protein therapeutics

基于 Python 的生物治疗药物实体精确质量计算工具

Over the past two decades, biotherapeutics have evolved to become a mainstay of the modern pharmaceutical industry. The SARS-CoV2 pandemic and other life-threatening conditions have further increased the need for the faster and broader development of biopharmaceutical molecules1,2,3.
The biotherapeutic molecular weight is critical for the identification of the molecule, in combination with other analytical assays. The intact and reduced subunit masses are used throughout the discovery and development lifecycles as part of control strategies aimed at maintaining the quality, as described in the QTPP (Quality Target Product Profile)4.
Analytical development in the biopharmaceutical industry relies heavily on mass measurements for intact mass analysis and deep characterization using peptide mapping or multi-attribute method (MAM) monitoring. At the center of these techniques utilizing modern mass spectrometry (MS) platforms is the ability to provide high-resolution accurate mass (HR/AM) measurements. Most HR/AM instruments yield mass accuracies in the range of 0.5-5 ppm, which scale with the mass range. The ability to measure masses accurately for intact large molecules enables the quick and confident identification of large-molecule therapeutics. As isotopic resolution cannot be attained using typical experimental conditions for large molecules (&#62;10 kDa), average masses must be calculated for comparison and identification5,6.
A typical intact or subunit protein mass spectrum represents the overall proteoform profile, which contains composite information on the various molecular forms resulting from post-translational modifications (PTM) and any primary structure differences, such as clips or sequence variants. The relatively easy and high-throughput nature of these measurements make them attractive for characterization and as in-process monitoring controls7,8. Data analysis for these experiments usually requires the user to define the search space for molecular forms (range of PTMs or other molecular forms). For glycosylated proteins, this search space is largely driven by glycoform heterogeneity. Combinations of multiple PTMs, disulfide bond configurations, and other variations along the primary structure make calculating all the possible molecular forms a tedious task. Therefore, the manual calculation of the possible molecular forms is a time and resource-consuming process with a high potential for human error.
Here, we present a mass calculation tool that was developed considering the most important features of biotherapeutic molecules, such as mAbs, bsAbs, ADCs, etc. The tool allows the easy incorporation of search-space variables for the consistent calculation of masses and elemental compositions. The modular nature of this tool will enable it to be further developed and applied to mass calculation and mass matching for other modalities.
The GUI module allows the user to specify the input for the mass calculation, as shown in Figure 1; specifically, the user enters single-letter amino acid sequences for light and heavy antibody chains. Common modifications for heavy-chain N-terminal cyclization and C-terminal lysine clipping are included as check boxes. Further, the chemical formula/elemental composition can be added/subtracted from these protein chains through the respective Chem Mod text box. This allows the user the flexibility to add an elemental composition that includes multiple post-translational modifications or a small-molecule payload in the case of an ADC. As most therapeutic mAbs are engineered to remove the glycosylation sites in the light chain, glycosylation in the light chain is left optional and can be specified using a check box on the GUI.
A typical variation on intact mass analysis for antibodies is a reduced subunit mass analysis, where the light chain is detached from the heavy chain by reducing the interchain disulfide bonds. Depending on the strength of the reducing agent used, the intrachain disulfide bonds may or may not be cleaved. The users have the flexibility of entering the total number of disulfide bonds depending on the IgG subtype or in case of a cysteine-conjugated ADC9.
The application calculates masses in a bottom-up manner, in which the elemental compositions are first calculated for the individual heavy chains and light chains. Next, heavy chain (HC) N-terminal cyclization Lys-clipping is accounted for by adjusting the calculated elemental compositions. Any specified chemical modifications are then applied to the heavy and/or light chains. Depending on the type of analysis and the disulfide-bond patterns specified by the user, the number of hydrogens is adjusted for the two polypeptide chains. The glycosylated HC and light chain (LC) (optional) masses are calculated based on the user&#39;s input. Finally, multiple HC and LC masses are combined, and the disulfide bond numbers are automatically updated for the intact mass calculation.
With larger molecules such as intact proteins, monoisotopic masses cannot be measured due to the additive mass defect when using mass spectrometers with typical resolving power. Instead, nominal or average masses are measured or reported5,10,11,12,13. The average elemental masses can vary based on the source used for the curated masses14,15. While the differences in elemental masses may be small, they can add up to significant values for large-molecule molecular weight calculations. The average elemental masses used by default in the software application are shown in Supplementary Table 1. For regulated environments like the biopharmaceutical research and development (R&#38;D) field, it is important to maintain consistent molecular masses because changes in masses may imply changes to the molecular entity during regulatory filings. To enable consistency in the use of elemental masses, a dictionary of elemental masses is included with the software tool as a comma-separated value (csv) text file: Element_Mass.csv (Supplementary Coding File 1). Similarly, a curated list of glycan compositions typically seen on mAbs is included: Glycan.csv (Supplementary Coding File 2). Both files are saved in the same folder location as an executable application and can be modified by the user to use a specific elemental mass list or glycan library.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65298/65298fig01.jpg" /> 
Figure 1: GUI interface for the mAbScale application. The GUI module allows the user to specify the input for the mass calculation. The user enters single-letter amino acid sequences for the light and heavy antibody chains. Common modifications for the heavy-chain N-terminal cyclization and C-terminal lysine clipping are included as check boxes. Chemical formulas/elemental compositions can be added/subtracted through the respective Chem Mod text box. <a href="https://www.jove.com/files/ftp_upload/65298/65298fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

作者聚焦：通过推出基于 Python 的桌面应用程序 mAbScale 推进生物治疗药物质量计算

用于基于抗体的治疗分子质量数计算的开源框架

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 MAPs, 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.MapScale 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.

Watch this Scientific Journal Video about Running the mAbscale Application to Calculate the Masses of Monoclonal Antibody-Based Protein Therapeutics at JoVE.com

Running the mAbscale Application to Calculate the Masses of Monoclonal Antibody-Based Protein Therapeutics  

运行 mAbscale 应用程序以计算基于单克隆抗体的蛋白质治疗药物的质量数

首先，通过双击可执行文件的图标打开软件应用程序。在 1 列下的文本框中输入重链和轻链序列，不带任何空格。对于双特异性抗体，在 2 列下的第二组文本框中添加额外的重链或轻链。对于具有相同重链和轻链的单克隆抗体，将这些框留空。如果这些重链端子变体适用，请选中末端端子环化和 C 端子剪裁复选框。使用氯化钙等元素组合物，将化学修饰物（包括抗体药物偶联物的接头和有效载荷）添加到重链和轻链化学修饰盒中。这些修饰将被添加到相应的蛋白质亚基或链中。在标有二硫键总数的文本框中指定蛋白质分子中二硫键的数量。根据还原程度，在未还原重链二硫化物文本框中输入未还原重链二硫键的数量，在未还原轻链二硫化物文本框中输入未还原轻链二硫键的数量。如果单克隆抗体轻链上存在糖基化，请选中“轻链被糖基化”复选框。单击“浏览”按钮为输出文件夹文本框选择输出文件夹，然后在“Excel 文件无扩展名”文本框中输入不带文件扩展名的输出文件名。然后单击“提交”按钮。屏幕上将出现质量计算报告创建消息，输出文件可以在用户指定的文件夹中找到。该应用程序将自动关闭。下表总结了各种单克隆抗体的化学成分、计算质量数、测量质量数和质量数误差。选择市售单克隆抗体标准品来代表FC区域具有相同重链、相同轻链和一个N-连接糖基化位点的常规单克隆抗体。还选择了具有额外轻链N-连接糖基化的单克隆抗体、双特异性单克隆抗体和抗体药物偶联单克隆抗体，以扩大应用范围。计算质量数与测量质量数之间的小质量误差在正常质量误差接受标准范围内，表明计算出的质量数是准确的。

running-mabscale-application-to-calculate-masses-monoclonal-antibody

Research

JoVE Journal

Biochemistry

Running the mAbscale Application to Calculate the Masses of Monoclonal Antibody-Based Protein Therapeutics

134 次观看.  GlaxoSmithKline. 首先，通过双击可执行文件的图标打开软件应用程序。在 1 列下的文本框中输入重链和轻链序列，不带任何空格。对于双特异性抗体，在 2 列下的第二组文本框中添加额外的重链或轻链。对于具有相同重链和轻链的单克隆抗体，将这些框留空。如果这些重链端子变体适用，请选中末端端子环化和 C 端子剪裁复选框。使用氯化钙等元素组合物，将化学修饰物（包括抗体药?...

视频: Running the mAbscale Application to Calculate the Masses of Monoclonal Antibody-Based Protein Therapeutics  

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

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers. 
We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.

This article describes the use of a software application, mAbScale, for the calculation of masses for monoclonal antibody-based protein therapeutics.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides ...

科研

生物化学

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 1 column without any spaces. For bispecific antibodies, add additional heavy or light chains in the second set of text boxes under the 2 column.Leave these boxes blank for monoclonal antibodies with identical heavy chains and light chains. Check the end terminal cyclization and C terminal clipping check boxes 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 check box. 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.