Protein Characterization and Analysis (ICH Q6B)
Protein analysis and characterization services, in line with the ICH Q6B Guidance, including protein structure analysis, physicochemical properties, biological activity, immunochemical properties and purity and impurities determination
Protein analysis and characterization to meet the challenges of drug development can be complex. Characterization of biologic 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 the development of a protein therapeutic, extensive analysis and characterization are required from an early stage in order to establish a well-characterized (specified) molecule and a good understanding of the protein’s structure, activity, immunochemical and physicochemical properties, purity/impurities and the impact of process changes on these factors. To support regulatory submission, the product should be compared with an appropriate reference standard, if available, and appropriately characterized in-house reference materials established.
Our protein analysis scientists provide characterization services in accordance with the ICH Q6B Guidance to ensure the quality and consistency of your product. With strengths in protein structure analysis including higher order structure, 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. We apply our expertise to 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 molecule-specific, strategic 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 glycoproteins, biosimilars, monoclonal antibodies, PEGylated proteins, antibody drug conjugates, bispecifics, and vaccines. Our laboratories are equipped with a broad technology base and our strengths in mass spectrometry (Orbitrap and QToF) analysis, in particular, allow the ability to provide high-resolution, accurate-mass (HRAM) data.
To support your submission, Intertek 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. Bringing quality and safety to life, we apply our Total Quality Assurance expertise to help you to meet and exceed quality, safety and regulatory standards for your biologic development programs.
Our scientists provide protein structural characterisation for the confirmation of identity and drives detailed understanding of your molecule’s structure from primary sequence through to higher order structure.
Amino-acid composition can be determine with our dedicated amino-acid analyser (AAA).
Our experts perform sequencing studies using a broad range of enzymatic digestion with high resolution accurate-mass spectrometry (Orbitrap, QToF) coupled with U(H)PLC technology.
Terminal Amino Acid Sequences
Confirmation of the amino- (N-terminal) and carboxy-terminal (C-terminal) amino acids is performed by MALDI-MS and high resolution mass spectrometry (Orbitrap, QToF) for 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 resolution mass spectrometry (Orbitrap, QToF) analysis. Once the methodology is established, we take the methods through validation for application in batch release or stability studies.
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 high resolution mass spectrometry (Orbitrap, QToF) 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 high resolution mass spectrometry (Orbitrap, QToF), 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 high resolution mass spectrometry (Orbitrap, QToF) following enzymatic digestion. Similar 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.
We determination the exact molecular weight of proteins using high resolution mass spectrometry (Orbitrap, QToF). We can also apply a range of other techniques such as size exclusion chromatography (SEC), SDS-polyacrylamide gel electrophoresis (under reducing and/or non-reducing conditions).
Isoform pattern and impurity studies are conducted using chromatography and or gel / capillary electrophoresis (PAGE, SDS-PAGE, IEF, CIEF / iCIEF, 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/ iCIEF), 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 higher-order structure is examined using far and near-UV circular dichroism (CD), nuclear magnetic resonance (NMR), infrared (FTIR), intrinsic fluorescence studies or ultraviolet-visible (UV-vis) spectroscopy (second derivative) spectroscopy. Our biophysical characterisation suite of technologies also includes protein aggregation studies through dynamic light scattering, SEC with multi-angle laser light scattering (MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC).
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.