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

बायोथेरेप्यूटिक संस्थाओं की सटीक द्रव्यमान गणना के लिए एक पायथन-आधारित उपकरण

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>

लेखक स्पॉटलाइट: mAbScale शुरू करके बायोथेरेप्यूटिक मास गणना को आगे बढ़ाना, एक पायथन-आधारित डेस्कटॉप एप्लिकेशन

एंटीबॉडी-आधारित चिकित्सीय अणुओं की बड़े पैमाने पर गणना के लिए एक ओपन-सोर्स फ्रेमवर्क

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  

मोनोक्लोनल एंटीबॉडी-आधारित प्रोटीन थेरेप्यूटिक्स के द्रव्यमान की गणना करने के लिए एमएबीस्केल एप्लिकेशन चलाना

शुरू करने के लिए, निष्पादन योग्य फ़ाइल के लिए आइकन पर डबल क्लिक करके सॉफ़्टवेयर एप्लिकेशन खोलें। बिना किसी रिक्त स्थान के 1 कॉलम के तहत टेक्स्ट बॉक्स में भारी श्रृंखला और हल्की श्रृंखला अनुक्रम दर्ज करें। द्विविशिष्ट एंटीबॉडी के लिए, 2 कॉलम के तहत टेक्स्ट बॉक्स के दूसरे सेट में अतिरिक्त भारी या हल्की चेन जोड़ें।इन बक्से को समान भारी चेन और हल्की चेन के साथ मोनोक्लोनल एंटीबॉडी के लिए खाली छोड़ दें। यदि ये भारी श्रृंखला टर्मिनल वेरिएंट लागू होते हैं तो अंतिम टर्मिनल साइक्लाइजेशन और सी टर्मिनल क्लिपिंग चेक बॉक्स की जांच करें। कैल्शियम क्लोराइड जैसे मौलिक रचनाओं का उपयोग करके भारी और हल्की श्रृंखला रासायनिक संशोधन बक्से में एंटीबॉडी दवा संयुग्म के लिए लिंकर और पेलोड सहित रासायनिक संशोधन जोड़ें।इन संशोधनों को संबंधित प्रोटीन सबयूनिट या श्रृंखला में जोड़ा जाएगा। डाइसल्फ़ाइड की कुल संख्या चिह्नित पाठ बॉक्स में प्रोटीन अणुओं में डाइसल्फ़ाइड बांड की संख्या निर्दिष्ट करें। कमी की सीमा के आधार पर अपरिवर्तित भारी श्रृंखला डाइसल्फ़ाइड टेक्स्ट बॉक्स में अपरिवर्तित भारी श्रृंखला डाइसल्फ़ाइड बांड की संख्या और अपरिवर्तित प्रकाश श्रृंखला डाइसल्फ़ाइड बॉन्ड की संख्या दर्ज करें।यदि मोनोक्लोनल एंटीबॉडी प्रकाश श्रृंखला पर ग्लाइकोसिलेशन मौजूद है, तो जांचें कि प्रकाश श्रृंखला ग्लाइकोसिलेटेड चेक बॉक्स है। आउटपुट फ़ोल्डर पाठ बॉक्स के लिए आउटपुट फ़ोल्डर का चयन करने के लिए ब्राउज़ करें बटन पर क्लिक करें और Excel फ़ाइल नो एक्सटेंशन पाठ बॉक्स में फ़ाइल एक्सटेंशन के बिना आउटपुट फ़ाइल नाम दर्ज करें. इसके बाद सबमिट बटन पर क्लिक करें।एक सामूहिक गणना रिपोर्ट बनाया गया संदेश स्क्रीन पर दिखाई देगा और आउटपुट फ़ाइल उपयोगकर्ता निर्दिष्ट फ़ोल्डर में पाई जा सकती है। एप्लिकेशन स्वचालित रूप से बंद हो जाएगा। विभिन्न मोनोक्लोनल एंटीबॉडी की रासायनिक रचनाओं, गणना किए गए द्रव्यमान, मापा द्रव्यमान और द्रव्यमान त्रुटियों को इस तालिका में सारांशित किया गया है।एफसी क्षेत्र में समान भारी श्रृंखलाओं, समान प्रकाश श्रृंखलाओं और एक एन-लिंक्ड ग्लाइकोसिलेशन साइट के साथ एक पारंपरिक मोनोक्लोनल एंटीबॉडी का प्रतिनिधित्व करने के लिए एक व्यावसायिक रूप से उपलब्ध मोनोक्लोनल एंटीबॉडी मानक का चयन किया गया था। एक अतिरिक्त प्रकाश श्रृंखला एन-लिंक्ड ग्लाइकोसिलेशन, एक द्विविशिष्ट मोनोक्लोनल एंटीबॉडी और एक एंटीबॉडी दवा संयुग्म मोनोक्लोनल एंटीबॉडी के साथ एक मोनोक्लोनल एंटीबॉडी को भी आवेदन के उपयोग को चौड़ा करने के लिए चुना गया था। गणना किए गए द्रव्यमान और मापा द्रव्यमान के बीच छोटी द्रव्यमान त्रुटियां सामान्य द्रव्यमान त्रुटि स्वीकृति मानदंड के भीतर थीं, यह सुझाव देते हुए कि गणना किए गए द्रव्यमान सटीक थे।

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 ...