Related Experiment Video
Updated: Jul 31, 2025
10:21
Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins
Published on: January 13, 2023
2.5K
A Spike-Control Approach that Evaluates High Resolution Mass Spectrometry-Based Sequence Variant Analytical Method
Jinhui Zhang1, Mack Shih2, Haoheng Yan3,4
1Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, USA. Jinhui.Zhang@fda.hhs.gov.
Pharmaceutical Research
|May 1, 2023
Summary
A novel spike-control method effectively validates analytical procedures for identifying and quantifying amino acid sequence variants (SVs) in biotherapeutics, ensuring product quality and safety throughout the product lifecycle.
Area of Science:
- Biopharmaceutical Analysis
- Protein Drug Product Quality Control
- Analytical Method Validation
Background:
- Amino acid sequence variants (SVs) are unintended substitutions in protein drugs, impacting heterogeneity, quality, safety, immunogenicity, and efficacy.
- Accurate analysis of SVs is crucial across the biotherapeutic product lifecycle, from early development to post-approval monitoring.
- Current SV analysis methods are complex, necessitating a systematic approach for validation.
Purpose of the Study:
- To introduce and evaluate a "spike-control" approach for assessing the capability of analytical procedures to reliably identify and quantitate SVs in biotherapeutics.
- To demonstrate the suitability of the spike-control method for validating SV analytical workflows.
Main Methods:
- A "spike-control" strategy was developed, using an FDA-approved monoclonal antibody (mAb) spiked with a highly homologous mAb to create artificial SVs.
- Spike-control samples underwent denaturation, reduction, alkylation, and digestion.
- Samples were analyzed using high-resolution Orbitrap mass spectrometry, with in silico analysis to identify differences representing SVs.
Main Results:
- Four artificial single amino acid sequence variants were reliably identified using the current analytical workflow.
- The analytical range, accuracy, and precision for SV quantitation were evaluated, showing suitability from 0.01% to 2%.
- The spike-control samples successfully demonstrated that the complete SV analytical procedure was fit for purpose.
Conclusions:
- The "spike-control" approach provides a robust method for validating analytical procedures used in SV identification and quantitation.
- This systematic evaluation ensures the reliability of SV analysis, critical for maintaining biotherapeutic product quality and safety.
- The validated workflow is suitable for identifying and quantifying SVs at pre-determined thresholds in biotherapeutics.
