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Peptide & Protein Characterization

Novatia provides routine LC-MS analysis of proteins/peptides along with 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.

Our routine LC-MS analysis of proteins/ peptides can be directly requested by using the “Submit a sample” button below. To inquire about our comprehensive sameness studies please use the “Contact Us” button below and select questions about NMR analysis.


Click on the blue highlighted topics below to explore further.

Impurities and degradants of peptide-based APIs can originate during synthesis or form over the shelf-life of the product (also see characterization of modifications below). Identification and quantification of these API-related impurities is an important part of verifying the purity and safety of drug substances and products. Novatia methods utilize state-of-the-art UPLC column technology and gradients to maximize the potential for separation of individual impurities from the API. In the case that impurities co-elute, we use high resolution MS data to provide reasonable estimates of the proportion of each species in a given UV peak. These methods allow for detection of impurities below 0.05% total LC-UV area% and quantitation of those present at levels greater than 0.1% total LC-UV area%.

Mass Spec analysis of a peptide or protein is an effective method to confirm or determine the primary sequence. For short peptides, a top-down MS/MS approach is a more direct method and is a common tool employed in the sequencing of peptides. MS/MS of peptides and proteins produce unique fragments along the peptide backbone which are used to identify the primary sequence.

LC-MS/MS analysis of liraglutide

Depending on the length of the peptide or protein, a bottom-up MS/MS approach may be required to provide sufficient sequence coverage. The main benefit of using a bottom-up MS/MS approach is the utilization of an enzymatic digestion to generate smaller peptide species that are ideal for MS/MS fragmentation. In cases such as liraglutide, a bottom-up MS/MS approach provides an orthogonal approach to obtaining sequence coverage from a top-down MS/MS approach.

A bottom-up sequencing approach (protein peptide mapping) can also be used to confirm the sequence of mAbs.

Peptides and proteins can contain a variety of modifications and their characterization can be important for a host of biological applications (ex: peptide API impurity profiling, see above). Novatia offers a diverse range of services to characterize modifications. These services include the identification of peptide API degradants and synthesis impurities, sample treatments to evaluate glycosylation, peptide mapping, and drug antibody ratio determination, among other routine services and custom projects. Below are two highlights that demonstrate the range of analytical methods offered by Novatia to characterize peptide/protein modifications.

Identification of Peptide API Related Isomeric Species by LC-MS

Most impurities can be  identified by LC-MS, however isomeric substitutions present a unique challenge as they cannot be identified by mass alone. Identification of API impurities containing isomeric amino acids often rely on searching for retention time (RT) shifts. However, depending on the sequence of the peptide containing the isomer, RT shifts may not be predictable. An example of this would be common peptide API isomers that form during the deamidation of aspartic acid or glutamic acid. Novatia has developed a bottom-up LC-MS/MS approach to differentiate these iso-amino acids (iso-aspartic acid and iso-glutamic acid) from their canonical counterparts based on a unique fragmentation pattern.

Drug Antibody Ratio (DAR) Determination by LC-MS

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.