Reference Standards to Support Quality of Synthetic Peptide Therapeutics

Abstract

Purpose: Peptides are an important class of therapeutics. Their quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength.

Objective: Discuss approaches to producing peptide reference standards, including vialing, lyophilization, analytical testing and stability studies.

Methods: Case studies are used to illustrate analytical approaches to characterize reference standards, including methods for value assignment, content uniformity, and identity testing. Methods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities.

Results: This report describes the analytical strategy used to establish peptide reference standard and illustrates how results from multiple labs are integrated to assign a value to the final lyophilized vial. A two-step process for value assignment is described, which uses a mass balance approach to assign a quantitative value to a bulk peptide material. The bulk material is then used as a standard to assign a final value to the vialed material. Testing to confirm peptide identity and to ensure consistency of the vialed material is also described. Considerations for addressing variability, identifying outliers, and implementing stability studies are also presented.

Conclusion: The methods and case studies described provide a benchmark for best practices in establishing the preparation, analytical testing, handling, and storage of peptide reference standards for the pharmaceutical industry. Some peptide features, such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization of the peptide reference standard.

Keywords: analytical characterization; content assignment; peptide pharmaceuticals; quality attributes; reference standards; stability testing.

Fig. 1

¹H/¹³C HSQC NMR Spectrum for Leuprolide Acetate

Fig. 2

MS Spectrum of Leuprolide Acetate Bulk Material. The high-resolution MS spectrum for [M + H] and [M + 2H] leuprolide ions agrees with expected mass

Fig. 3

MS/MS Analysis of Leuprolide Acetate Bulk Material. The high-resolution MS/MS spectrum for [M + 2H] ion agrees with expected fragments for leuprolide

Fig. 4

Variability of Value Assignment for Desmopressin Acetate Across Laboratories. Peptide mass content results for each of 10 preparations of the vialed material are shown for each laboratory to illustrate the variability both within and across laboratories (note that Lab L11 only assayed three preparations). Analysis of variance (ANOVA) showed that lab L9 results had statistically significant differences when compared to the mean of the other five labs

Fig. 5

Accelerated Stability Studies of (A) Gonadorelin and (B) Bivalirudin. This figure shows the results of the accelerated stability testing where peptides were subjected to thermal stress at multiple temperatures. While gonadorelin was highly stable, bivalirudin was more susceptible to thermal stress and showed significant degradation at higher temperatures