Polyethylene glycol, or PEG, is a synthetic polymer that is attached to certain biopharmaceuticals during production. Examples of some PEGylated drugs include pegfilgrastim (a PEGylated form of GCSF), interferons and certolizumab pegol. PEG can be synthesized such that all molecules have the same length, or it can be made as a polymer of varying lengths. PEG polymers can also have different numbers of branches.
Why are some proteins PEGylated?
PEG is usually attached to proteins through the amine group side chains of amino acids (usually Lysine) in order to suppress potential immunogenicity or prolong the half-life over the native protein. The mechanism for these desired outcomes relies on the fact that the addition of PEG makes it more difficult for the immune system to detect the drug and therefore clear it. PEG can also be also used to increase the solubility of hydrophobic protein drugs.
PEGylated proteins present unique challenges to analytical assessment and structural characterization. For example, heterogeneity in the PEG moiety can result in complex profiles from particular characterization methods such as intact mass analysis or Edman sequencing, where PEG may block to certain reactions when it is located at the N-terminus of the protein.For some of these investigations it may be necessary to chemically de-PEGylate prior to analysis.
Furthermore, the chemical process used to couple the PEG unit to the protein may result in adverse chemical reactions occurring to other parts of the protein such as oxidation or deamidation events, disulfide bridge scrambling or modifications to glycosylation, therefore it is particularly important for PEGylated molecules that a thorough characterization of the protein be undertaken.
Characterization of PEGylated Biopharmaceuticals
BioPharmaSpec scientists have been characterizing PEGylated proteins for over three decades and can work with you to devise the best structural and physicochemical characterization strategies for your development plans.
In this case study, a BioPharmaSpec client requested NMR analysis of their PEG-GCSF product and in particular requested that BioPharmaSpec purify the PEGylated N-terminal peptide to provide an assessment of the linkage between PEG/Linker and the N-terminal Methionine residue.
In this case study, a BioPharmaSpec client requested the determination of the extinction coefficient of their PEG-GCSF product. Understanding the extinction coefficient of your biopharmaceutical is essential for considerations of the dosage and concentration of your product. It is not sufficient to rely on theoretical (software derived) or experimental (using amino acid analysis) estimations, which can have up to 15% error from the true value.
Regulatory Requirements for the Characterization of PEGylated Proteins
Whether the product of interest is a new product or a biosimilar (e.g. PEGylated GCSF), structural and physico-chemical characterization are a crucial part of biopharmaceutical product development. The expectations of the EMA and US FDA, in terms of structural characterization, are outlined in ICH Topic Q6B.
ICH Topic Q6B provides examples of characterization assays which might be considered for structural characterization and confirmation, and evaluation of physicochemical properties of the desired product, drug substance and/or drug product. The guideline recognizes that new analytical technology and modifications to existing technology are continuously being developed and should be utilized when appropriate.
Regulatory guideline documents recommend that the following characterization methods are performed:
- Primary (de novo) Amino Acid Sequence
- Amino Acid Composition and extinction coefficient
- Terminal Amino Acid Sequence
- Peptide Map and Post Translational Modifications (PTMs)
- Disulfide Bridges
- Carbohydrate Structure
- Identity and Purity
- Molecular Weight
- Isoform Pattern
- Liquid Chromatographic Patterns
- Secondary and Tertiary Structure
Product Specific Technical Considerations
Particular attention should be paid to the following structural elements which are important and challenging when analyzing PEGylated proteins:
1. Site(s) of PEGylation and level of occupancy
Is the PEG unit attached where it should be and how efficient was the PEGylation process?
2. Disulfide bridges
Are the disulphide bridges correct or has scrambling taken place?
3. Product- and process-related impurities
Has the PEGylation reaction resulted in modifications to the protein itself and what other components are present in the sample as a result of the subsequent purification process?
Has the PEGylation reaction caused deamidation of the protein and where has this taken place on the protein chain?
Has the PEGylation reaction caused oxidation of the protein and where has this taken place on the protein chain?
Please contact our scientists if you would like to discuss appropriate characterization strategies for your PEGylated biopharmaceutical.