Protein analysis in line with ICH Q6B Guidance for characterisation of protein structure, physicochemical properties, biological activity, immunochemical properties, purity and impurities
Protein analysis can present significant challenges. Characterisation of protein or biological therapeutics includes analysis of protein structure, physicochemical properties, biological activity, immunochemical properties, purity and impurities, as described in the ICH Q6B Guidance, Specifications: Test Procedures and Acceptance Criteria for Biotechnological /Biological Products.
During development of a protein, extensive analysis and characterisation is required to establish a well-characterised (specified) molecule to achieve a good understanding of the protein’s structure, physicochemical properties, biological activity, immunochemical properties, purity and impurities and the impact of significant process changes on these. To support regulatory submission, the product should be compared with an appropriate reference standard, if available, and appropriately characterised in-house reference materials established.
Our protein analysis scientists provide characterisation and testing services in accordance with the requirements of the ICH Q6B Guidance to ensure the quality and consistency of your product. With strengths in protein structure analysis, physiochemical property determination, biological and functional assays, product- and process-related impurity testing we can help you to achieve a "well characterized" protein drug substance or drug product. Through experienced protein analysis we can support your product development, in-process testing, identity confirmation, comparability, stability testing and batch release testing.
We work with our clients to design and deliver bespoke and molecule specific strategic protein analytical programs, designed to optimise the data required to determine or confirm identity, explore patterns of heterogeneity and demonstrate consistency in quality of the drug substance or drug product such as proteins glyco-proteins, biosimilars, monoclonal antibodies, PEGylated proteins, antibody drug conjugates, bispecifics, and vaccines.
To support your submission, we can provide data to Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP), as appropriate, with comparison against the most suitable reference standard, if available.
Our protein analysis scientists provide structural characterisation for the confirmation of identity and drives detailed understanding of your molecule’s structure from the primary sequence of amino acids through to extent of heterogeneity and higher order structure.
Amino-acid sequence and composition
The distribution of amino acids in the antibody product can be determined via a number of approaches. Our experts then perform sequencing studies using a broad range of enzymatic or chemical digestion with LC-MSMS mass spectrometry analysis.
Terminal amino acid sequences
Confirmation of the amino- (N-terminal) and carboxy-terminal (C-terminal) amino acids is performed by LC-MSMS specifically with the aims of product identification and to establish homogeneity, where understanding the type and extent of modifications at either termini, is a fundamental aspect of product quality control.
Intertek conduct selective fragmentation of protein into discrete peptides by enzyme or chemical digestion followed by high-performance liquid chromatography and electrospray mass spectrometry analysis and /or MALDI-TOF MS. Once the methodology is established we take the methods through validation and application in batch release or stability assessments.
Disulphide bridging Mapping (S-S bridge)
Where cysteine residues are present in the molecule, our scientists perform a qualitative/semi-quantitative assessment of the position and extent of expected and mismatched disulphide bridges by extended LC-MSMS peptide mapping studies, MALDI-TOF or electrospray MS and colorimetric tests for free sulfhydryl groups.
Carbohydrate or Glycosylation Structure
Glycosylation studies are designed product specific, however, these typically include determination of the levels of neutral and amino monosaccharides as well as sialic acids, assessment of glycoform distribution and glycan structure elucidation. Multiple technologies are applied in these determination including selective enzymatic cleavage and MALDI-TOF Mass spectrometry HPLC, HILIC, IEX or CE-LIF, to provide the level of structural information required.
Post Translational Modification Studies (PTM)
Our post translational modification analysis experts apply a strategic approach to PTM analysis during early development phases to help you to establish product acceptance criteria and as part of structural characterization studies and comparability programs, stability studies or quality control testing.
Position of Conjugation
For antibody drug conjugates (ADCs) our scientists determine the position of attachment of the toxin to the linker and subsequently protein, also known as the sites of conjugation. This is typically achieved using LC-MS and LC-MS/MS following enzymatic digestion. The same logic methods can also be applied to establishing the position of PEGylation and other conjugated forms.
Our scientists design and deliver physicochemical property analytical programs in line with appendix 6.1.2 in the ICH Q6B guidelines Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Our programs are designed to optimise the data required to determine or confirm identity, explore patterns of heterogeneity and demonstrate consistency in quality of the drug substance. We are adept at method development and phase-appropriate validation where required.
Our scientists apply a range of techniques to determine molecular weight such as size exclusion chromatography (SEC), SDS-polyacrylamide gel electrophoresis (under reducing and/or non-reducing conditions) and mass spectrometry. We offer the determination of exact molecular weight of proteins or other biologics in a drug sample using LC ESI-MS with comparison against the expected molecular weight of the drug substance.
Isoform pattern and impurity studies are conducted using chromatography and or gel / capillary electrophoresis (PAGE, SDS-PAGE, IEF, CE, HPLC).
Extinction Coefficient (or Molar Absorptivity)
Our protein scientists accurately determine the extinction coefficient using UV/visible absorbance on solutions of the product which have a known protein content. We quantify the concentration of protein in solution using amino acid compositional analysis, either by using an amino acid analyser or UPLC instrumental approaches.
Electrophoretic patterns and data on identity, homogeneity and purity can be obtained by polyacrylamide gel electrophoresis, capillary isoelectric focusing (CIEF), SDS-polyacrylamide gel electrophoresis, Western-blot or capillary electrophoresis.
Liquid Chromatographic Patterns
Using size exclusion chromatography (SEC), reverse-phase liquid chromatography (RP-HPLC), ion exchange liquid chromatography (IEX), or UPLC approaches we generate chromatographic patterns and data on the identity, homogeneity, and purity.
The ultraviolet and visible absorption spectra are determined as appropriate. The higher-order structure of the product is examined using circular dichroism (CD), nuclear magnetic resonance (NMR), infrared (FTIR) or fluorescence spectroscopy.
We provide a range of total protein quantification methods, including European Pharmacopoeia (EP) and United States Pharmacopeia (USP) methods. Colorimetric assays such as the Bradford or bicinchoninic acid (BCA) assays measure UV-light absorbance and can be used to calculate protein concentration from the absorbance measurement, once the extinction coefficient (molar absorptivity) has been accurately established. We also provide quantitative amino acid analysis by high performance liquid chromatography (HPLC) or ion chromatography (IC).
Our protein analysis scientists determine process impurities such as those related to cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or chromatographic media used in purification, solvents and buffer components. To address product related impurity analysis our team utilise a wide range of technology (chromatographic, electrophoretic, mass spectrometry, spectroscopy) to determine truncated or modified variants, degradation products or conjugated forms. We have a great deal of experience in determining the presence of aggregates and degradation products.
We are experienced in method development, method transfer, method validation of tailored bioassays to GMP standards and conducting routine potency assays using cell migration, ligand binding, ELISA, cell signalling, cell proliferation and inhibition of proliferation, binding assays or competitive assays approaches.
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