Peptide-based Complex API and Product Sameness

Background:

In 2017, the Food and Drug Administration (FDA) issued its ANDA Guidance for Certain Highly Purified Synthetic Peptide Drug Products that Refer to Listed Drugs of rDNA Origin. This guidance outlines the requirements for ANDA submissions regarding complex active pharmaceutical ingredient (API) sameness relative to established products.

The complex API of a generic drug can be compared both qualitatively and quantitatively for sameness relative to reference listed drug (RLD) samples. This can be done using mass spectrometry (MS) to determine primary sequences, and 1D and 2D NMR spectroscopy to determine higher order structure (HOS).

Novatia as a Complex API and Product Sameness Research Partner:

Novatia has experience in applying the FDA guidelines to characterize complex API sameness for several clients. An example of characterization of two different APIs and formulated drug products (DP) is shown below in Figure 1 and Figure 2. Glucagon, the active ingredient in GlucaGen (an emergency medicine for hypoglycemia) is compared to liraglutide, the active ingredient in Victoza (a non-insulin medication used to lower blood sugar levels in patients with Type II Diabetes) for sameness.

NMR Characterization for Complex API’s

For peptides and proteins, the chemical shifts of backbone nitrogens and protons are very sensitive to primary, secondary, and tertiary structure. Small changes in the local environment can affect these backbone chemical shifts. Spectra of two common complex APIs (glucagon and liraglutide) are shown below. The pattern of chemical shifts in the amide N-H bond region of a ¹H spectrum (6-11 ppm, see black boxes) is considered a fingerprint for each peptide, allowing for rapid qualitative comparison of samples for solution-structure sameness.

**Figure 1:** TOP: Stacked 1D ¹H NMR of glucagon and liraglutide illustrating how peptides of similar molecular weight and amino acid count can easily be distinguished.
BOTTOM: Overlay expansion of 1D ¹H NMR of glucagon and liraglutide in which fingerprint backbone proton resonances are observed. (liraglutide DS: 6mg/mL in 90:10 buffered H₂O/D₂O; glucagon DS: 2 mg/mL in 90:10 H₂O/D₂O).

Thus, the 1D ¹H NMR can differentiate between two complex peptide APIs or be used to profile multiple lots of a specific complex API target for HOS sameness. However, the addition of inactive ingredients to the Drug Substance (DS) in the DP formulation can result in increased complexity, obscuring key regions of the 1D ¹H NMR spectrum. In this example, adding lactose to the glucagon DS introduces non-API signals in the aliphatic/anomeric region of the ¹H spectra of the DP (Figure 2 red spectra). Other excipients such as phenol, found in liraglutide DP, overlap with the signal of the amide fingerprint region (Figure 2 blue spectra).

**Figure 2:** The stacked spectra above illustrate the complexity in the 1D ¹H NMR of glucagon and liraglutide DP formulation once excipients were added. (glucagon DP Formulation: 1 mg/mL in 90:10 H₂O/D₂O, 49 mg lactose, 12 mg/ml glycerin, pH=2.5-3.5 with H₃PO₄; liraglutide DP Formulation: 6 mg/mL in 90:10 H₂O/D₂O, 1.42mg disodium phosphate dihydrate, 14mg propylene glycol, 5.5 mg phenol, pH=8.1).

This increased spectral complexity due to addition of excipients in the DP formulations may present difficulties in determining complex product sameness by 1D NMR spectroscopy as shown above. This complexity can be addressed by applying 2D NMR with natural abundance ¹⁵N. Using 2D NMR, the fingerprint amide proton and nitrogen of each amino acid of a complex peptide API can be observed without interference from excipients. Figure 3 shows an overlap of the ¹H – ¹⁵N HMQC NMR spectra of the liraglutide DS and DP Formulation in which the phenol resonances are not visible, as this experiment only detects protons bound to nitrogens.

**Figure 3:** An overlay of the 2D ¹H –¹⁵N HMQC NMR spectra of the liraglutide DS and DP shows that the presence of excipients is not observed in the fingerprint region. Small changes in chemical shift are a result of changes in the solvent matrix between the DS and excipients in the DP. Resonances in the circled region for liraglutide DP are due to arginine side chain groups likely visible due to the lower pH of the DP sample versus that of the DS sample. (*liraglutide DS: 6mg/mL in 90:10 buffered H₂O/D₂O*; *liraglutide DP Formulation: 6 mg/mL in 90:10 H₂O/D₂O, 1.42mg disodium phosphate dihydrate, 14mg propylene glycol, 5.5 mg phenol, pH=8.1*).

Novatia is uniquely positioned to assist research organizations in process development and NMR characterization for regulatory filing of complex APIs. We also offer combined services with our MS team for further characterization. Contact us to see how Novatia’s NMR/MS teams can assist your complex API needs.