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Synthetic peptide therapeutics of increasing complexity are being evaluated as a modality to access targets that are difficult to drug with small molecules. Analytical challenges associated with the development of peptide drug substances include resolving impurities that are chemically similar to the desired peptide API, and also involve the added need for biophysical characterization. This talk covers recent advances in analytical technologies such as capillary electrophoresis coupled with mass spectrometry and ion mobility – mass spectrometry for analytical characterization of peptides beyond purity and molecular weight determinations.
Over the last decade mass spectrometry (MS) has been increasingly implemented in QC laboratories to support e.g. research, quantification, release analysis or the control of LC methods.
Hereby, coupling of LC-UV and MS analysis has been proven particularly useful as it offers an orthogonal way of detection by linking UV responses with their associated molecular masses. Thus, co-elution of non-isobaric compounds can be discovered rapidly by LC-MS and, since the underlying principle is discrimination by mass, MS/MS may be applied for supplementary structural investigations. In turn, because MS is constantly developing into a more widely used standard QC method, software and instrument compliance is another crucial aspect of MS-related analyses that needs the attention of users and vendors.
We developed a semi-automated LC-MS data analysis method to facilitate identification of impurities, that are co-eluting with UV-Peaks of interest and are thus not detectable by UHPLC analysis alone. Additionally, identification and absolute quantification of identified impurities has been accomplished using tandem mass spectrometry and spiking experiments, respectively.
LC-MS data acquisition was performed applying standard parameters and a MS acquisition frequency of 2 Hz. After integration of the UV Peak of interest a custom made DataAnalysis script automatically reads out the extracted ion chromatograms (EIC’s) of all m/z values over a previously defined intensity threshold. All generated EIC’s are subsequently listed by the software and evaluated for congruency. EIC’s that show congruent trends with the main compound are discarded as “artefacts” generated due to e.g. in-source CID, while EIC’s diverging from the main compounds EIC are further analyzed to confirm or rule out the presence of an impurity. We also show that the utilization of a 2D map (m/z vs. retention time) is a very useful tool to facilitate detection of co-eluting compounds, especially when corresponding EIC’s are inconclusive or not precisely generated.
Furthermore, we also want to highlight from a routine QC lab point of view, the needs regarding MS-software usability in a regulated environment. It seems that while the technical advancement of mass spectrometers proceeded rapidly, corresponding software development often lags behind, particularly in terms of usability, and compliance-related functionalities are merely applied “on-top” of existing core software rather than being an integral part of the software development process right from the start. Consequently, we want MS vendors to understand the current bottle necks when performing MS in a GxP setting and where small changes or add-ons would greatly facilitate routine lab processes.
The presentation will describe the strategy for analytical method development for both commercial and development peptides. Various challenges posed by peptides are presented in the form of case studies. QbD is built into the method development phase by using a DoE approach to ensure rapid method validation.
The notable expansion of peptide and oligo therapeutics development in the last two decades has provided a robust pipeline that should deliver numerous approvals during the remainder of the 2010s. This changing landscape of the pharmaceutical drug pipeline from predominantly small molecules to an even distribution between small and large molecules has resulted in changes of analytical workflows that were typically used in the past for bioanalysis. Traditional approaches to quantify these molecules in biological matrices have utilized immunoassay approaches that can be time inefficient, have limited analytical ranges and lack assay specificity. The lack of assay specificity can result in not accurately distinguishing large metabolites from the full-length compound of interest. The improvements in sample preparation technologies, chromatographic systems and mass spectrometers over the last decade have meant that LC-MS/MS approaches to peptide and protein quantification are feasible and can overcome the problems associated with quantification by immunoassay. Unfortunately, there is no one-size-fits-all approach for measuring these varied compounds. The goal of method development is an assay that can quantify levels of protein, peptides and oligo therapeutics with high confidence to ensure a successful biotherapeutic drug development. An overview of the current bioanalytical method development approaches will be given including associated case studies related to peptides and oligo therapeutic development.
Peptide therapeutics present unique advantages and challenges in drug development. Key to any drug development program is adequate characterization and understanding of the pharmacokinetics of the drug, namely, absorption, distribution, metabolism and excretion. Additionally, nonclinical safety evaluation is critical in setting the dose/exposure for early clinical studies (Phase 1/2). The davunetide drug development program will be used as a case study to illustrate how pharmacokinetic and toxicology data is collected and interpreted.