Protein Characterization Service
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Proteins are complex molecules with three-dimensional structures and their unique structure is responsible for their biological activity. The amino acid sequence is referred to as the primary structure of a protein. The hydrogen bonds between amino acids of the primary structure result in secondary structures, typically in the form of alpha helices and pleated sheets. In the tertiary protein structure, hydrogen bonds, ionic, hydrophobic, and disulfide bridges are formed between regions of the secondary structure.
Proteins are composed of amino acid chains that interact to form quaternary structures. By using orthogonal analytical techniques, it is important to characterize all of these levels of protein structure. At CBA, we have state of art 21 CFR compliant equipment to fully characterize the primary and higher order structure of protein molecules.
Proteins are biomolecules made up of chains of amino acids. Characterizing proteins requires different analytical tools to examine their primary, secondary, tertiary, and quaternary structures. At CBA we offer characterization services aligned to ICH Q6B to determine the physicochemical properties, process & product related impurities and biological properties.
We have in house methods which have been validated as per ICH Q2 (R1) and where ever applicable pharmacopeial methods are used. At CBA, we will help you characterize your molecule better for USFDA, EMEA, CDSCO and other regulatory agencies submissions.
Following are few services currently being offered at CBA.
Protein Intact Mass Analysis using HRMS
Mass analysis of intact proteins using high resolution mass spectrometry is a rapid method for confirming the identity and the primary structure of proteins. Mass analysis can provide information for evaluating and comparing the isoforms, glycoforms, and molecule wide modifications associated with a protein. The analysis can readily compare manufacturing batches or can be used to compare a biosimilar to the innovator. Measurement of molecule weight is a requirement of the regulatory guidelines too. We offer intact mass analysis under non-reducing, reducing, N-deglycosylated conditions.
Peptide Mapping using HRMS
Peptide mapping is a useful technique for primary structure analysis of proteins. As per ICH Q6B, peptide mapping is important to test for structural characterization and confirmation. The peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing, or mass spectrometry.
Peptide mapping of the drug substance or drug product using an appropriately validated procedure is a method that is frequently used to confirm desired product structure for lot release purposes. Peptide mapping can also help in identifying Product-related impurities including degradation products. Peptide mapping is a useful tool to establish similarity between innovator and biosimilar product. Peptide mapping has been widely accepted as an identity test for biotherapeutics in the QC lab. We offer peptide mapping service using UPLC as front end and bottom-up approach (Enzyme digestion).
Host Cell Protein Impurity Identification using HRMS
Host Cell proteins are process related impurities which can be immunogenic and hence are required to be quantitated. As per FDA, the limit for HCPs in final drug product is 1-100 ppm. ELISA is a gold standard for HCP analysis, but it heavily relies on special immunoreagents. In contrast, HRMS an orthogonal method for HCP analysis does not depends on special reagents and offers sensitive detection of peptides accurately and faster. We offer HCP analysis by MS analysis using Nano LC as front end and bottom-up approach (Enzyme digestion).
Post Translational Modifications using HRMS
Common PTMs observed in protein molecules are glycosylation, oxidation, deamidation, and phosphorylation. PTMs can have a potential effect on the stability, efficacy and safety profile of the proteins. Hence, getting the correct information about these PTMs is critical. At CBA, we are using chromatographic and high-resolution mass spectrometry tools to understand and provide a comprehensive characterization of all the PTMs.
N-glycan analysis using HRMS
Glycosylation is a critical quality attribute that affects the physicochemical, pharmacological and pharmacokinetic profile of the protein. Glycan analysis commonly refers to N-Glycan analysis. N-glycans can be characterized as released glycans, glycopeptides, glycosylated protein subunits, and intact glycoproteins. The most common among these is released glycan analysis. At CBA, we are equipped with High Resolution Mass Spectrometry (HRMS) as a powerful tool for glycans profiling and UPLC based quantitation of released glycans.
Disulfide linkage analysis
Disulfide bonds are important in protein folding and have significant impact on structural and functional properties of protein. Disulfide bond is formed by covalent bonding between sulphur atoms of Cysteine residues. If the protein contains cysteine residues, then the disulphide bonds analysis is required for physicochemical characterization of the molecule. Bottom-up MS approach is used and enzyme digested reduced and non-reduced samples are analyzed and compared. Multiple enzyme digestion and deglycosylation may be required for some molecules.
Secondary and tertiary structure analysis using far-UV CD Spectroscopy
Circular dichroism is an established characterization technique that can be successfully used in biopharmaceutical process development for detecting conformational changes or assessing the comparability of test samples with references. Equality of spectra can be assessed either visually or by spectral deconvolution, extracting from the spectra a set of numerical values that represent the secondary structure content of a given protein. In the “Far-UV” spectral region (190-250 nm) peptide bonds produce CD signals that are sensitive to the overall secondary structure of the protein.
By analyzing the CD spectra of the Far-UV region, a defined assessment of the components of the protein secondary structure can be made. In the “near-UV” spectral region (250-350 nm) aromatic amino acids and disulfide bonds produce CD signals that are sensitive to the overall tertiary structure of the protein. By analyzing the CD spectra of the near-UV region, a good assessment of the tertiary structure of the protein sample can be made. At CBA, we offer secondary and tertiary structure analysis by CD spectroscopy. We also offer temperature-dependent CD analysis.
Tertiary Structure Analysis using Fluorescence Spectroscopy
The amino acid tryptophan has the strongest fluorescence quantum yield of the amino acids found in proteins. The rest either do not fluoresce or fluoresce weakly. Therefore “Intrinsic Protein Fluorescence” usually refers to the fluorescence emission of the tryptophan amino acids. In a hydrophobic environment (buried within the core of the protein), Tyr and Trp have a high quantum yield and therefore a high fluorescence intensity. In contrast in a hydrophilic environment (exposed to solvent) their quantum yield decreases leading to low fluorescence intensity. For Trp residue, there is strong stoke shifts dependent on the solvent, meaning that the maximum emission wavelength of Trp will differ depending on the Trp environment. At CBA, we measure intrinsic tryptophan fluorescence to determine the tertiary structure using fluorescence spectroscopy.
Higher Order Structure Analysis using DSC
Protein maintains its higher order structure using multiple ionic, non-ionic, hydrophobic, and other interactions between its subunits. However, at higher temperatures when these interactions are disrupted protein unfolds and eventually denatures. Using Differential Scanning Calorimetry, which collects information on heat released when protein is unfolding, its melting temperature (a temperature where protein denatures) can be measured. Typically, the melting temperature of a protein is its unique property and can be used as a comparative parameter to establish the integrity of biotherapeutic products. At CBA, we measure the apparent Tm of a protein using Nano-DSC in order to confirm the higher order structure.
Aggregate/Multimers Analysis using SEC-MALS
Aggregates in drug products can adversely affect the patients and induce an immunogenic response. As per ICH Q6B – aggregates include dimers and higher multiples of the desired product. These are generally resolved from the desired product and product-related substances, and quantitated by appropriate analytical procedures. SEC-MALS combines a multi-angle light scattering detector with size-exclusion chromatography. It can determine molecular weight from 200 g/mol to 1 billion g/mol and hence is a routinely used method for aggregate analysis. At CBA, we offer aggregation analysis by SEC-MALS technique.
Protein-Protein Interaction Analysis using SPR
Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein-protein interactions, specifically affinity and binding kinetics. It is a gold standard for early efficacy measurements and is a widely used assay for regulatory submissions. The measurements are in real-time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the ligand, on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the analyte, over the surface containing the ligand. The binding is monitored by subsequent changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. At CBA, we offer binding kinetics and affinity analysis using a SPR system.
Protein analysis by PAGE
ICH Q6B requires electrophoretic patterns for the analysis of Biopharmaceuticals. PAGE technique would be used to carry out protein identification, size determination, impurity analysis, and aggregation analysis.
Liquid Chromatography analysis
Chromatographic methods can be used for protein quantitation, purity, identity, glycan, impurity, sialic acid, monosaccharide, and stability analysis. At CBA, UPLC system with fluorescence detector is dedicated for amino acid composition, purity, and glycan workflows.
Amino acid composition analysis
Amino acid composition analysis is a classical protein analysis method. It is a complex technique, comprising three steps, hydrolysis of the substrate, labeling of hydrolyzed amino acids with a suitable fluorophore and chromatographic separation, and detection of the fluorophore-labeled amino acid residues. The obtained chromatograms of samples are compared with the chromatograms of standard fluorophore-labeled amino acids. The comparison allows the identification and quantification of amino acids present in the sample.
Charge variants analysis by cIEF
Charge variants of a biopharma products are protein molecular species that have different overall charge. Analysis of charge variants is a key requirement of ICH Q6B. These might arise due to the degradation, changes in processes, or PTMs. Isoelectric point (pI) for a protein is a pH at which protein molecules carry no net charge. Capillary Isoelectric focussing (cIEF) is a widely used method for charge variant analysis. It allows the determination of the apparent pI of mAbs, levels of acidic, main, and basic mAbs variants as well as impurities.
Cell based assays help determine CQAs like efficacy, safety and immunogenicity. Proliferation & cytotoxicity, apoptosis, ADCC and CDC assays are being offered at CBA. We have defined different assay platforms for different molecules.