Rapid Top-Down Analysis of the NISTmAb antibody
Others | 2020 | BrukerInstrumentation
Top-down analysis of intact proteins offers rapid and direct verification of the primary sequence and terminal modifications of biotherapeutic antibodies. This approach reduces the time and complexity associated with traditional peptide mapping, enabling high-throughput screening and robust quality control for monoclonal antibodies.
The study demonstrates a streamlined MALDI top-down workflow for rapid sequencing of NISTmAb light and heavy chains. It focuses on verifying N- and C-terminal modifications, assessing sequence integrity, and improving laboratory productivity when processing multiple protein therapeutics.
The workflow begins with chemical reduction of the antibody using guanidine hydrochloride and dithiothreitol, followed by buffer exchange and concentration via centrifugal filter units. Reduced chains are spotted onto a specialized MALDI plate, washed, and co-crystallized with a dihydroxybenzoic acid matrix. Data acquisition is performed on a MALDI mass spectrometer without the need for liquid chromatography separation.
Top-down sequencing of the NISTmAb light chain yielded approximately 70 % sequence coverage, confirming the expected amino acid sequence with no unexpected modifications. Heavy chain analysis identified N-terminal pyroglutamate modification and confirmed removal of C-terminal lysine residues. The method provided clear N- and C-terminal readouts within minutes, illustrating both speed and accuracy for intact antibody chain verification.
Advancements may include coupling MALDI top-down with higher resolution detectors to extend coverage beyond 25 kDa, development of automated sample handling platforms, and application to other complex biotherapeutics such as fusion proteins and antibody-drug conjugates. Integration of complementary fragmentation techniques could further improve sequence completeness.
The presented MALDI top-down sequencing approach delivers a fast, robust, and user-friendly solution for verifying antibody chain sequences and terminal modifications. Its high throughput and simplicity make it well suited for quality control and sequence confirmation in biopharmaceutical research and manufacturing.
MALDI, LC/TOF, LC/MS
IndustriesClinical Research
ManufacturerBruker
Summary
Importance of Top-Down Antibody Sequencing
Top-down analysis of intact proteins offers rapid and direct verification of the primary sequence and terminal modifications of biotherapeutic antibodies. This approach reduces the time and complexity associated with traditional peptide mapping, enabling high-throughput screening and robust quality control for monoclonal antibodies.
Objectives and Overview
The study demonstrates a streamlined MALDI top-down workflow for rapid sequencing of NISTmAb light and heavy chains. It focuses on verifying N- and C-terminal modifications, assessing sequence integrity, and improving laboratory productivity when processing multiple protein therapeutics.
Methodology
The workflow begins with chemical reduction of the antibody using guanidine hydrochloride and dithiothreitol, followed by buffer exchange and concentration via centrifugal filter units. Reduced chains are spotted onto a specialized MALDI plate, washed, and co-crystallized with a dihydroxybenzoic acid matrix. Data acquisition is performed on a MALDI mass spectrometer without the need for liquid chromatography separation.
Used Instrumentation
- rapifleX MALDI mass spectrometer (Bruker Daltonics)
- BigAnchor sample plates (Bruker Daltonics)
- Amicon Ultra-0.5 centrifugal filter units, 10 kDa cutoff
- BioPharma Compass 3.1 software for data processing
Main Results and Discussion
Top-down sequencing of the NISTmAb light chain yielded approximately 70 % sequence coverage, confirming the expected amino acid sequence with no unexpected modifications. Heavy chain analysis identified N-terminal pyroglutamate modification and confirmed removal of C-terminal lysine residues. The method provided clear N- and C-terminal readouts within minutes, illustrating both speed and accuracy for intact antibody chain verification.
Benefits and Practical Applications
- Substantial time savings over LC-MS peptide mapping workflows
- High-throughput capability for routine quality control of therapeutic antibodies
- Direct detection of terminal modifications, sequence variants, and truncations
- Minimal sample preparation and elimination of chromatographic steps
Future Trends and Opportunities
Advancements may include coupling MALDI top-down with higher resolution detectors to extend coverage beyond 25 kDa, development of automated sample handling platforms, and application to other complex biotherapeutics such as fusion proteins and antibody-drug conjugates. Integration of complementary fragmentation techniques could further improve sequence completeness.
Conclusion
The presented MALDI top-down sequencing approach delivers a fast, robust, and user-friendly solution for verifying antibody chain sequences and terminal modifications. Its high throughput and simplicity make it well suited for quality control and sequence confirmation in biopharmaceutical research and manufacturing.
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
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