Porton Biopharma has a long history of pharmaceutical development and manufacturing at Porton Down. Both Erwinase® and Anthrax Vaccine were developed at this site, as well as many other products, such as Dysport® and Voraxase® which have subsequently been out-licensed to other companies.
Porton Biopharma are developing a product that is a complex mixture of different proteins. They contacted BioPharmaSpec to discuss the possibility of developing a method using mass-spectrometry to identify the proteins present and quantify the amounts of these detected proteins in different formulations (from ng to µg/mL) of the product.
Technical Considerations- Method Optimization
BioPharmaSpec has in-house methods for protein identification and relative quantitation in a wide-range of protein mixtures. Our scientists were tasked with optimizing a method for analysis that was reproducible and sensitive enough to detect dilute protein material, whilst minimizing handling of the client’s product.
The optimization was performed using a single representative sample for analysis. The in-house methodology comprises:
- Concentration followed by proteolytic digestion of the products to release peptides for analysis.
- Analysis of the products of digestion using LC-MS/MS (and/or MSe) together with Q-TOF methods.
- Assessment of the obtained data against the host cell protein sequence database to create a spectral library of peptides from proteins in the product and identify the proteins present.
The data obtained from this initial optimization also allowed our scientists to identify peptide markers for the proteins detected. An estimate of the amount of each detected protein present was provided by performing further analysis following spiking of a representative product with three known proteins in known amounts.
Quantitation was then performed, comparing the responses obtained from peptides derived from the known amounts of the spiked proteins, to peptides derived from each individual protein detected in the product (using “hi-3” quantitation methodology) to gauge the relative amounts of proteins generated by proteolytic cleavage.
“BioPharmaSpec was able to get a method up and running very quickly and we started using it immediately for development studies. The method recently helped us to identify an important change in our product, which we confirmed using ELISA, and it has now become a crucial part of our in-process development.” – Innovation Lead– Porton Biopharma Ltd.