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Questions and Answers
What is the purpose of using purified polyclonal antibodies in RIA or ELISA approaches?
What is the purpose of using purified polyclonal antibodies in RIA or ELISA approaches?
What is a limitation of amino acid analysis in biopharmaceutical analysis?
What is a limitation of amino acid analysis in biopharmaceutical analysis?
What is the molecular mass limit for peptides or small polypeptides that can be studied using an automated amino acid analyzer?
What is the molecular mass limit for peptides or small polypeptides that can be studied using an automated amino acid analyzer?
Why is amino acid analysis not considered a complete test for identifying a protein?
Why is amino acid analysis not considered a complete test for identifying a protein?
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What is the purpose of collecting serum from animals in the production of polyclonal antibodies?
What is the purpose of collecting serum from animals in the production of polyclonal antibodies?
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What is peptide mapping used for in protein identification?
What is peptide mapping used for in protein identification?
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What is a disadvantage of amino acid analysis in detecting contaminants in the final product?
What is a disadvantage of amino acid analysis in detecting contaminants in the final product?
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What type of antibodies are used in RIA or ELISA approaches to detect contaminants in the final product?
What type of antibodies are used in RIA or ELISA approaches to detect contaminants in the final product?
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What is the purpose of performing multiple tests, including amino acid analysis, peptide mapping, and N-terminal sequencing, in protein identification?
What is the purpose of performing multiple tests, including amino acid analysis, peptide mapping, and N-terminal sequencing, in protein identification?
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Why is it necessary to combine multiple tests, including amino acid analysis, peptide mapping, and N-terminal sequencing, in protein identification?
Why is it necessary to combine multiple tests, including amino acid analysis, peptide mapping, and N-terminal sequencing, in protein identification?
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Study Notes
Two-Dimensional Electrophoresis
- Separates proteins based on molecular weight and isoelectric point, enhancing detection of impurities.
- Captures product homogeneity by revealing a single protein band.
- Assesses product stability over its shelf life.
SDS-PAGE Limitations
- Only separates proteins by mass; cannot distinguish contaminants of equal weight.
Alternative Purity Determination Techniques
- HPLC effectively analyzes both high and low molecular mass proteins.
- Electrospray mass spectrometry ionizes proteins for detailed mass analysis through high electric fields.
Potency Testing
- Expressed as "units of activity per vial or dose" or in milligrams.
- Bioassays: Tests the functionality of proteins by reconstituting with water and assessing activity, using model systems (e.g., viral infections in mice).
- All bioassays are comparative against a standard.
Contaminants in Final Products
- Residual proteins may have similar properties (size, isoelectric point) to the protein of interest, potentially remaining in the final product.
- These residuals can be immunogenic, especially upon repeated administration.
- Covalent modifications may result in biologically inactive or modified pharmacokinetic forms, necessitating their removal.
Downstream Processing Techniques
- Removal relies on exploiting differences between modified and target proteins, such as size discrepancies (achieved via size-exclusion chromatography).
Detection Techniques for Protein-Based Impurities
- Non-denaturing electrophoresis, SDS gel electrophoresis, capillary electrophoresis (expensive), peptide mapping, isoelectric focusing, HPLC, and mass spectrometry.
- Mass spectrometry is the most precise method for determining protein molecular mass.
Antibody Testing for Contaminants
- Serum from animals can be analyzed using purified polyclonal antibodies in RIA or ELISA to check for cell line-derived contaminants.
Protein Identity Verification
- Methods include amino acid analysis, peptide mapping, N-terminal sequencing, and analysis of secondary/tertiary structures.
- Amino Acid Analysis: Involves using an automated analyzer; limits include potential destruction of amino acids during hydrolysis and moderate sensitivity.
Overall Testing Strategy
- No single test confirms protein identity; multiple methods are combined for comprehensive analysis.
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Description
This quiz covers the advantages of two-dimensional electrophoresis over SDS-PAGE in detecting protein-based product impurities, including its ability to separate proteins based on molecular weight and isoelectric point.
