A peptide is usually considered to be any polymer of 40 or less amino acids. This means they are small biopharmaceuticals with less structural complexity than, for example, monoclonal antibodies. Peptide drugs can be chemically synthesized or recombinantly produced using a cell line such as E.coli. Peptide products may have a homogenous sequence, where all molecules in the product have an identical amino acid sequence, or they may be composed of peptides with heterogenous sequences. A heterogenous peptide product is a mixture of peptides with different sequences, such as glatiramer acetate.

Peptide Structure

Peptide products can have complex shapes that are highly dependent on the correct amino acid sequence. For this reason, peptides often present unique and difficult structural characterization challenges.

Peptide structure: Linaclotide

One example of this would be the confirmation of the disulfide bridge structure in Linaclotide. Linaclotide is only 14 amino acids long but contains three disulfide bridges when it is correctly produced. The below schematic shows the 14 amino acids, using single letter code, and the positions of the three disulfide bonds.

Linaclotide has a very tightly bound structure, due to the high number of disulfide bonds relative to the size of the molecule. This creates challenges for structural characterization because the structure must be fragmented in order to allow mass spectrometric analysis.

An example of a challenge in the structural characterization of peptides is the assessment of fatty acid acylation in Liraglutide, namely confirmation of location and identity of the acyl chain. The nature of the peptide amino acid sequence can often place constraints on the procedures that can be applied for structural characterisation. Frequently the simpler procedures are not possible to employ because, for example, amino acids that would commonly be used as points of enzyme proteolysis, are not present. In these cases experience of handling these types of molecules gives an understanding of the strategies that need to be employed in order to characterise them.

Structural Characterization of Peptides

The regulators have seen a rapid increase in the number of applications submitted for peptide drug products driven mainly by the significant number of generic peptide product submissions. Characterization approaches differ significantly according to the peptide product being analyzed.

ICH guideline Q6A addresses the regulatory requirements for developing synthetic peptides of low molecular weight. Based on this guideline, identification testing should discriminate between compounds of closely related structures that are likely to be present. In the Linaclotide example above, this would include identification of the molecules with mismatched (also known as scrambled) disulfide bridges.

Identification tests should be specific for the drug substance. Therefore, identification by a single chromatographic retention time alone is not regarded as being specific. However, the use of two chromatographic procedures, where the separation is based on different principles or the use of a combination of tests into a single procedure, such as HPLC/UV diode array, HPLC/MS, or GC/MS is generally acceptable.

For peptide products with a single defined (homogenous) sequence, BioPharmaSpec has developed methods to provide in-depth information for the product and any product-related impurities.

For generics of heterogeneous peptide drug products (i.e. those that vary in amino acid length and sequence, such as glatiramer acetate), the requirement is to demonstrate that the active ingredients in the generic are comparable to the Reference Product. This type of analysis requires high-end analytical characterization and a comparability assessment using statistically significant data.

Structural and Physicochemical Characterization Methods for Peptides

  1. Intact molecular weight analysis including an assessment for product-related impurities
  2. Primary amino acid sequence confirmation
  3. N-and C-terminal sequence analysis and confirmation of intactness
  4. Peptide mapping following proteolytic digestion (if required)
  5. Disulfide bridge analysis (for Cysteine containing peptides)
  6. Analysis of post translational modifications, if appropriate
  7. Amino acid analysis to provide amino acid molar ratios and peptide content
  8. Chromatographic analysis (RP-HPLC, IEX and SEC)
  9. IsoElectricFocusing (icIEF)
  10. Aggregation analysis (SEC-MALS and SV-AUC)
  11. Secondary and tertiary structure analysis (spectroscopic profiling; 1D and 2D NMR, Circular Dichroism (CD), Fourier Transform-Infra Red (FT-IR) and Fluorescence as appropriate)
  12. Assessment of process-related impurities