Successful and rapid analytical method development plays a key role in ensuring the development of robust processes to produce high-quality products. When standard or predefined methods fail because of buffer components or because proteins do not behave as expected, solutions can be found when the team has the knowledge and flexibility to explore the use of alternate methods.

 CASE STUDY 1 

Process Development and Analytical Development for a Challenging Therapeutic Molecule (Homodimer Fusion Protein)

For a protein of interest, there were no analytical methods to measure the amount and form of the refolded protein that was expressed in Escherichia coli as an insoluble product extracted from inclusion bodies and then refolded into a noncovalent dimer. In order to develop and optimize the process to produce a sufficient quantity of material for initial toxicity studies, the process development group needed a robust analytical method that could be used to assess and quantify the properly folded protein.

The solution

Several standard methods were first evaluated (including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)), but these methods did not provide the necessary level of resolution to separate the dimer from the other components.

In this case, the poor resolution could be due to the complex refolding buffer matrix. A review of the literature on these types of products revealed that another type of high-performance liquid chromatography (HPLC) — ion-exchange chromatography (IEX) — might provide a solution.

Analytical scientists developed an IEX-based analytical method to quantify the refolded molecule in the post-refold sample. The IEX assay was used to distinguish the charge differences of the correctly refolded dimer material from the improperly folded/aggregated species and other host cell proteins, monomers and fragments in the refold material. In-process quantification was performed based on using the standard curve generated from final bulk material.

This assay, which could accurately measure the properly refolded protein versus other species, was critical to process development efforts to improve refolding efficiency, to scaling up the process to pilot scale and to providing the required quantity and quality of material for toxicology studies.

 CASE STUDY 2 

Protein Measurement in the Presence of Detergents

Protein quantification of the final bulk  material is critical to process and analytical assay development, and is also required to perform other analytical testing, like enzyme-linked immunosorbent assays (ELISA) and SDS-PACE assay. For a particular protein, the inclusion of components in the buffers, such as dithiothreitol (DTT) and Tween-20 detergent, to solubilize the protein during purification process development caused the front-line traditional assays to fail to work. The A280 method did not work due to the presence of oxidized DTT, which absorbs at the 280 nm wavelength. A bicinchoninic acid assay (BCA) failed due to DTT interference, and a Bradford assay failed due to Tween interference. The Pierce 660 kit-based assay was tolerant to DTT and Tween; however, the assay runs at acidic pH, which possibly caused precipitation of the product. Developing a protein assay that is tolerant to detergents was required.

The solution

A size-exclusion column (SEC)-based method was explored and eventually developed that can quantify protein concentration in the presence of the interfering detergents without precipitating the protein. The SEC method is easy and robust, and the method is isocratic compared with other HPLC-based methods requiring use of a gradient of buffer. The protein concentration is determined on the basis of a bovine serum albumin (BSA) standard curve with application of the corrected extinction coefficient value.  Even with the SEC-based method, Tween caused significant assay interference at high concentrations; therefore, the Tween concentration needed to be tracked. SEC could not be used to determine Tween concentration, likely due to oxidation over the 30-minute assay time, so a colorimetric assay was developed to track and control Tween 20 detergent through the purification process.

Summary

Analytical tools are the key to understanding and optimizing any process, and having the experience and means to explore alternative methods allows the analytical scientist to rapidly meet the demands of any project. Where appropriate, established methods based on existing standard operating procedures (SOPs) and protocols are relied upon. When those established methods fail, custom solutions are developed to meet the needs of the product and project.

The ability to use alternative methods to solve problems is the key to developing novel protein molecules. These two case studies demonstrate the value brought to the process when scientists with extensive training are given the freedom to apply their knowledge and identify new approaches to the challenges we face.  Each challenge builds on the existing foundation and improves the traditional methods to increase efficiency, productivity and quality. 

Grifols’ Experience Leveraged for Customer Molecules

Scientific understanding of the protein chemistry and biology, knowledge of current analytical tools, and disciplined problem solving approaches, lead to successful development of novel analytic methods.

The process and analytical development teams at Grifols Recombinant Protein CDMO Services, which include legacy staff from Chiron and Novartis, have expertise in diagnostic antigen and therapeutic protein development.

We are focused on providing support for our customers and ensuring that we have the process and analytical method development resources needed to meet all deliverables. Open and transparent communication, a high degree of flexibility and extensive experience with recombinant protein development are fundamental to Grifols’ development strategy.