Skip to main content

Peptide & Protein Characterization

Novatia provides comprehensive sameness studies; including LC-MS impurity profiling, NMR-based structural studies, and aggregation studies to support the development of peptide-based biosimilars. Our knowledge and experience with international regulatory landscapes enables us to help clients troubleshoot a wide scope of pharmaceutical problems from early development to navigating ANDA submission requirements for complex APIs.


Click on the blue highlighted topics below to explore further.

Detailed paragraph LC-MS profiling of peptide APIs, drug products, or drug substance impurities. Example: liraglutide

LC-MS or LC-MS/MS of intact or digested material​

DAR calculation can be determined following mAb analysis under standard antibody treatments such as reduction, Deglycosylated only, Deglycosylated and reduction as well as intact (for more information on sample treatment options, follow the mAb MS link).

The sample data and calculated DAR shown below are that of MSQC4-AF488, and MSQC8, antibody-drug conjugate mimics, consisting of the MSQC4 standard mAb with dansyl fluorophores attached to lysine or cysteine residues, respectively. Analysis examples include intact and pre-treated samples under either denaturing or native LC conditions*. More sample data can be found here.

*Denatured LC conditions expose samples to acidic (pH~2) and organic solvents during separation. Native conditions expose samples to aqueous salt buffers (pH~6-7) to favor maintaining stable protein fold.

Analysis of reduced and PNGase F treated ADC

The heavy and light drug-conjugated protein chains elute in two distinct peaks, following reduction of cysteine disulfide bridges and removal of N-linked glycans.

Below are the baseline subtracted ESI and deconvoluted spectra under the 5.480 minute peak. The deconvoluted mass spectrum demonstrates the ability of ProMass to accurately calculate drug-conjugate states of the heterogeneous MSQC4-AF488 heavy chain, with no observed mass artifacts. The DAR for the heavy chain of the MSQC4-AF488 is 3.7.

The baseline subtracted ESI and deconvoluted spectra under the 5.180 minute peak are shown below. The DAR for the light chain of the MSQC4-AF488 is 1.7.

The overall DAR for the reduced and PNGase F treated MSQC4-AF488 sample shown is 5.4.

For questions about our MS analysis of ADCs and DAR calculations, email ms@enovatia.com.

The figure shows a overlay of the 2D 1H –15N HMQC NMR spectra of Native (red contours) and SS scrambled (green contours) Bovine Pancreatic Ribonuclease A where cross peaks between directly bonded nitrogens and hydrogens (protons) can be observed. NMR chemical shifts are highly dependent and characteristic of structural environment. For proteins, the chemical shifts of nitrogens and protons are very sensitive to secondary and tertiary structure such that small changes in the local environment can affect the position of cross peaks in the spectrum. NMR spectra like these are useful for comparing HOS of biologics and biosimilars as the pattern of cross peaks can be considered as fingerprint allowing for rapid qualitative comparison of samples for three dimensional solution structure similarity.

With the recent boom of complex API based therapeutics coming to market, the Food and Drug Agency (FDA) has recently issued a guidance for Synthetic Peptides and a draft guidance regarding Sameness Evaluations of Complex APIs. Within these guidances, the agency recommends that “the applicant applies orthogonal analytical methods to characterize the Oligomer/Aggregation States.” Oligomer/Aggregation States can be characterized by a variety of orthogonal methods: Size Exclusion Chromatography (SEC-UV-MS), Dynamic Light Scattering (DLS), and Diffusion Ordered Spectroscopy (DOSY).

Recent advances in the field have revolved around the comparison of DLS and DOSY. One publication, by scientists at the Agency, suggests that DLS is more suitable than DOSY to determine whether aggregates that could cause immunogenicity are present in a sample. This is due to the higher sensitivity DLS has for evaluating higher molecular weight compounds.

Novatia has experience in applying DLS and its orthogonal methods for characterizing aggregation states of peptides. Shown below is an example of a DLS autocorrelation function for a lot of Ozempic. In addition to the translational diffusion coefficient (Dt), DLS evaluates the hydrodynamic radius (Rh), %Mass, and %Polydispersity (%Pd) for each autocorrelation. Furthermore, the %Pd characterizes the oligomeric makeup of a specific signal. Such as, whether the species is present as a single conformer or whether there are other unresolved oligomers present. In the case below, a %Pd between 15-30% corresponds to a species that consists of a combination of mainly monomer with a small population of oligomers.

Figure 1: Autocorrelation function of a lot of Ozempic. Autocorrelation 2 was determined to be both an effect of the formulation buffer and insignificant due to a %Mass < 0.1%.

Novatia has assisted research organizations with aggregation characterization for regulatory filing of complex APIs. We offer combined services with our MS team and NMR Team for further characterization. Contact us to see how our team can assist your aggregation characterization needs.