Name: Running the mAbscale Application to Calculate the Masses of Monoclonal Antibody-Based Protein Therapeutics
Uploaded: 2023-06-16T13:20:00
Description: 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

כלי מבוסס Python לחישוב מסה מדויק של ישויות ביותרפיות

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>

Author Spotlight: Advanceancing Biotherapeutic Mass Calculation by Introducing mAbScale, יישום שולחן עבודה מבוסס Python

מסגרת קוד פתוח לחישוב מסה של מולקולות טיפוליות מבוססות נוגדנים

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  

הפעלת יישום mAbscale לחישוב המסות של טיפולים חלבוניים מבוססי נוגדנים חד-שבטיים

כדי להתחיל, פתח את יישום התוכנה על ידי לחיצה כפולה על הסמל של קובץ הפעלה. הזן את רצפי השרשרת הכבדה והשרשרת הקלה בתיבות הטקסט מתחת לעמודה 1 ללא רווחים. עבור נוגדנים דו-ספציפיים, הוסף שרשראות כבדות או קלות נוספות בקבוצה השנייה של תיבות הטקסט מתחת לעמודה 2.השאירו קופסאות אלה ריקות עבור נוגדנים חד שבטיים עם שרשראות כבדות זהות ושרשראות קלות. בדוק את תיבות הסימון מחזוריות מסוף קצה וחיתוך מסוף C אם גרסאות מסוף שרשרת כבדות אלה ישימות. הוסף שינויים כימיים כולל מקשר ומטען עבור תרופות נוגדנים מצומדות לתיבות שינוי כימיות כבדות וקלות שרשרת באמצעות הרכבי יסודות כגון סידן כלורי.שינויים אלה יתווספו לתת-היחידה או לשרשרת החלבונים המתאימים. ציין את מספר הקשרים הדיסולפידים במולקולות החלבון, בתיבת הטקסט המסומנת כמספר הכולל של דיסולפידים. הזן את מספר הקשרים דיסולפידים של שרשרת כבדה לא מופחתת בתיבת הטקסט דיסולפידים של שרשרת כבדה לא מופחתת ואת מספר הקשרים דיסולפידים של שרשרת קלה לא מופחתת בתיבת הטקסט דיסולפידים של שרשרת קלה לא מופחתת, בהתאם למידת ההפחתה.אם גליקוזילציה קיימת בשרשרת האור של נוגדנים חד-שבטיים (monoclonal antibodies), סמן את תיבת הסימון light chain is glycosylated. לחץ על לדפדף כפתור לבחירת תיקיית פלט עבור תיבת הטקסט של תיקיית הפלט והזן את שם קובץ הפלט ללא סיומת קובץ לתיבת הטקסט קובץ Excel ללא סיומת. לאחר מכן לחץ על שלח לחצן.הודעה שנוצרה דוח חישוב המוני תופיע על המסך וניתן למצוא את קובץ הפלט בתיקייה המיועדת למשתמש. האפליקציה תיסגר באופן אוטומטי. ההרכבים הכימיים, המסות מחושבות, המסות הנמדדות וטעויות המסה של נוגדנים חד שבטיים שונים מסוכמים בטבלה זו.תקן נוגדנים חד-שבטיים זמין מסחרית נבחר כדי לייצג נוגדן חד-שבטי קונבנציונלי עם שרשראות כבדות זהות, שרשראות קלות זהות ואתר גליקוזילציה N-linked אחד באזור FC. כמו כן נבחרו נוגדן חד שבטי עם גליקוזילציה N-linked נוספת בשרשרת קלה, נוגדן חד-שבטי דו-ספציפי ונוגדן נוגדן מצומד נוגדן חד-שבטי להרחבת השימוש ביישום. טעויות המסה הקטנות בין המסות המחושבות למסות הנמדדות היו בתוך הקריטריונים הרגילים לקבלת שגיאות מסה, דבר המצביע על כך שהמסות המחושבות היו מדויקות.

Research

JoVE Journal

Biochemistry

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 ...