Post-translational modifications
Glycosylation can account for a great deal of a protein's heterogeneity, depending on how heavily glycosylated the protein
is. The population of sugar units attached to the individual glycosylation sites on any protein will depend on the host cell
type used, with cells derived from different species of animals, from plants, or from microbes producing different constellations
of sugar chains. For this reason, FDA recommends using the same type of cell to produce a biosimilar as was used for the innovator
wherever possible. Even within a single cell culture, however, the same polypeptide will be produced in a number of different
glycoforms.
Because glycosylation can have significant effects on a protein's efficacy, stability in circulation, or immunogenicity, it
is a parameter that needs to be well characterized and well controlled. At a minimum, the carbohydrate content of the protein
(neutral sugars, amino sugars and sialic acids) should be determined, in addition to the structure of the carbohydrate chains,
the oligosaccharide pattern (antennary profile), and the glycosylation site(s) of the polypeptide chain, explains.
"There are many dimensions to glycans—you can analyze to the level of one antigenic epitope, or perform a detailed analysis,
depending on the critical features of your protein." says Pauline Rudd, principal investigator at the National Institute for
Bioprocessing Research and Training (NIBRT) in Dublin, Ireland, explains. FDA, she says, is interested in glycosylation variants
that are known to produce immunogenicity in humans, for example, xylose or fucose from plant-derived proteins. But they will
have different concerns for different proteins. In epoetins, for instance, terminal sialylation has been shown to affect stability
in circulation, so for this type of product, the agency will expect that sialylation will be thoroughly described. Quantitative
technologies are essential and hydrophilic interaction liquid chromatography-based technologies that separate glycans on the
basis of lipophilicity, meet this criterion. Coupled with experiemntal databases such as NIBRT's Glycobase 3+ and exoglycosidase
digestions they provide robust tools. Confirmation of structure by an orthogonal technology is always required. Mass spectrometry
which, although only semi-quantitative, is an important tool as it separates glycan pools on the basis of mass and importantly
can be used to examine fine structural details. Whatever the level of analysis, she says, glycosylation patterns must be reproducible.
The developer defines limits, and the product must be within limits for every batch.
Although glycans are a critical feature of many biological products, they aren't everything, explains Rudd. There are 200
potential posttranslational modifications, including c-terminal clipping, disulfide bond formation, glycation, and deamidation.
Correct disulphide bond formation is crucial for achieving the proper three dimensional structure of the protein, but cysteine
residues can sometimes mispair so the disulphide bonds scramble. Rudd believes that more and more, tertiary structure analysis
(i.e., analysis of 3D-structure) will be required.
Orthogonal and overlapping techniques
When developing a biosimilar, physicochemical characterization is carried out to determine the complete sequence of the innovator
plus any post-translational modifications, and to evaluate the quality attributes and purity of the product. A side-by-side
comparison of the innovator and a proposed biosimilar will require extensive data on both the primary structure and higher
order structure of both molecules using a variety of techniques. Guidance on the choice of appropriate analytical methods
can be obtained from the ICH Q6B guideline (5). A single method may provide information on multiple protein attributes. For
instance, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) can provide information on size, presence of
aggregates, and the variability in disulfide bond formation, while isoelectric focusing can provide information on isoform
pattern that may reflect differences in glycosylation or deamidation.
The exact characterization strategy will depend on the individual molecule, but should include methods to compare size, charge,
and shape of the molecules. One of the most versatile and essential methods that can be used is mass spectrometry (MS), according
to Greer. MS can provide information on intact molecular weight, structure confirmation via mass mapping techniques, sequence
using MS/MS and identification on disulphide bridging, heterogeneity and posttranslational modifications, including glycosylation.
A host of additional techniques will also be required, particularly to compare the secondary and tertiary structure of the
molecules. In this respect, circular dichroism is a useful technique to measure how the protein is folded.
The process and the product
For biologics, the cell type in which the protein is produced, the exact conditions of cell culture, and the methods used
in downstream processing can all affect the disposition of the final product. A recent review of the effects of cell-culture
conditions on glycosylation found that nearly every aspect of cell culture could be demonstrated to have an effect, including
composition of the cell-culture medium, dissolved oxygen concentration, bioreactor pH, carbon dioxide partial pressure, temperature,
shear stress, and manufacturing mode (i.e., fed-batch versus perfusion) (6). Scale-up can also affect glycosylation of the
product, requiring characterization at multiple points during process development and scale up. First determining which glycosylation
attributes are critical to the function of the protein, then choosing analytical methods that optimally measure those attributes,
rather than trying a more scattershot approach may streamline the process (7).
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