The importance of characterization
BioPharm: Why is it important to characterize amino-acid sequence and carbohydrate structure?
NIBRT: Characterization of the amino-acid sequence--normally using LC-MS-based approaches, either top-down or peptide-centric bottom-up methods--is important to verify that the product produced is that as expected from the engineered gene sequence. Data from such experiments facilitate the determination and comparison of the experimental versus the predicted mass of the product. Deviations are indicative of post-translational or other modifications. Such modifications, if present, can then be localized when using peptide-centric LC-MS methods, or sequence variants can also be verified at the peptide level. Data from such studies can then be used to evaluate whether the presence of a particular modification is important with regard to the structure-function relationship of the molecule, or whether for safety or efficacy purposes, it may be necessary to engineer out the modified amino acid.
Similarly for carbohydrates, characterization of the glycosylation present with regard to monosaccharide analysis and structural characterization of the N- and O-linked oligosaccharides present is
important to ensure that the desired glycosylation is present and that potentially immunogenic epitopes are absent.
Characterization of the glycosylation present on recombinant proteins is particularly important as the glycans attached to the molecule can modulate the stability and ability of the molecule to elicit its desired effector response, particularly in the case of monoclonal antibodies (1). Because glycosylation is cell-line specific and also affected by the environmental conditions that a cell finds itself in, it is necessary to routinely characterize the oligosaccharides present to make sure that the expressed protein is being produced with consistent and reproducible glycosylation. Furthermore, characterization of the glycosylation present is important to verify the presence or the absence of potentially immunogenic epitopes such as galactose-α1-3-linked galactose motifs and the nonhuman monosaccharide N-glycolyl neuraminic acid.
Characterization of glycosylation is still a significant analytical challenge although recent advances in analytical instrumentation and separation chemistries have benefited the field considerably. Glycans are traditionally analyzed using either liquid-phase separation techniques such as liquid chromatography or capillary electrophoresis with optical or fluorescence detection or mass spectrometry. As oligosaccharides lack inherent chromophores or fluorophores, they must be derivatized to facilitate detection, fluorescent reagents, such as 2-amino-benzamide, 2-aminobenzoic acid, 2-aminopyridie or 2-aminoacridone, have been widely reported as derivatization reagents used in glycan analysis. When using capillary electrophoresis, charged fluorescent labels such as 1-aminopyrene-3,6,8-trisulfonic acid (APTS) or 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) are used to impart electrophoretic mobility to the labeled oligosaccharides and also to facilitate highly sensitive laser induced fluorescence detection.
Mass spectrometry (MS) has also been widely used for glycan analysis, either using matrix-assisted laser-desorption ionization (MALDI) or electrospray ionization (ESI). A caveat with the use of MS is its inability to distinguish isobaric monosaccharide residues or isomeric oligosaccharides; therefore, it is more a compositional analysis. MS/MS based methods can facilitate glycan sequencing when performed on [M+H]+ ions formed during positive ionization. MS/MS of [M-H]- ions formed when using negative ionization is considerably more informative as it can provide diagnostic ions that facilitate the deduction of linkage and positional analysis of the oligosaccharides. There have also been significant advances in associated informatics platforms for the annotation of both LC and MS data in recent years.
Furthermore, the issues of glycan micro- and macroheterogeneity must also be considered. Performing a global glycosylation screen informs us of the identity and relative abundance of the types of oligosaccharides attached to the molecule. However, combination with glycoproteomics is often necessary to identify the sub-populations of glycans present at each glycosylation site.The combination of analytical techniques in an orthogonal manner is recommended to impart confidence to the analytical data. It is not recommended to analyze the glycosylation using a signal technique; generally, a minimum of two approaches is recommended.
Quintiles: The amino-acid sequence of a biosimilar molecule is one of the starting points in determining similarity to the originator medicine, with the draft guidance from FDA implying that a biosimilar molecule needs to have the same amino-acid sequence as the originator medicine. Some proteins, such as monoclonal antibodies, are glycosylated (i.e., they have carbohydrate molecules attached to them). The extent and the exact structure of carbohydrates attached to a protein may affect its binding to its target receptor, its clearance from the body, and potentially, its immunogenicity. The manufacturing process (e.g., cell line used) can influence the exact nature and extent of glycosylation of the protein, and thus potentially, its activity and safety. It is not possible to definitively determine the structure of a large protein, such as a monoclonal antibody of approximately 150,000 Da, using currently available analytical techniques. This means that confirming similarity of two proteins requires the use of a wide range of analytical techniques.
Functional binding of molecules can be tested using microarray analysis. This technique enables the biosimilar developer to test the binding of the protein to a large number of targets to determine binding and eliminate potential cross reactivity. In many cases, this functional binding may be adequate if the effect of the medicine is simply to neutralize its target. Where the protein-based medicine acts through some cellular signaling pathway, there are tools for assessing these modes of action such as cell-based kinase assays.