Mechanisms for Protein Aggregation and Stabilization in Solution, during Freezing and Drying, and at Interfaces

AKA, Lessons Learned: Application of Mechanistic Insights to Solving Practical Protein Aggregation Problems

Dear Colleagues,

I am starting an effort to provide training to formulation and process development scientists in areas of fundamental mechanisms for protein aggregation and stabilization. The main title given above is for a lecture that covers several key areas. I would be happy to present the talk to groups of researchers and host discussions on the topics.

One example of the practical applications comes from a quiz that is part of the talk. Read the two cases described below. Then, choose the correct quiz answer based on mechanistic understanding of the impacts of excipients (such as sucrose) on protein stability and thermodynamic properties.

Two cases with monoclonal antibodies. For both, the bulk drug substances appeared cloudy after freeze-thawing.  (These are based on real-world examples.)

Case 1. Upon thawing the bulk drug substance, the solution was very cloudy, due to protein precipitation. The precipitate could be collected by centrifugation, immediately after thawing. Infrared spectra of the centrifuged precipitate indicated that there was only native secondary structure in the protein molecules in the precipitate. After an uncentrifuged sample was held at room temperature for 2-8 hours, the precipitates dissolved and the protein molecules went back into solution. 

Case 2. Upon thawing the bulk drug substance, the solution was very cloudy, due to protein precipitation. After the sample was held at room temperature for 2-8 hours, the precipitates settled, but did not go back into solution. Infrared spectra of the centrifuged precipitate indicated that there was extensive intermolecular beta sheet structure between the protein molecules in the aggregates. 

Quiz. In which case(s) would the inclusion of 0.3 M sucrose reduce the amount of precipitation; that is,  inclusion of the sugar in the bulk drug substance solution would be an effective formulation strategy? Why?

a) Case 1 

b) Case 2

c) Both cases

d) Neither case

e) No way to know without hiring of consultants and further, extensive "root cause analysis"