(368bv) Instrument Development to Rapidly Screen Lyophilized Protein Stability

Overview: The objective of my PhD work is to develop instruments to rapidly screen excipients of lyophilized (freeze-dried) proteins for long-term stabilizing capabilities. Currently, long-term protein stability studies require that the lyophilized protein be stored for several months, reconstituted, and tested regularly for degradation. With this work, formulation screening is performed directly in the solid phase with novel optical techniques. The presented techniques (low-frequency Raman spectroscopy and optical Kerr effect spectroscopy) show promise for reducing formulation screening from several months to a few minutes.

Research Interests: After earning my PhD, I am highly interested in applying my skills in instrument development and analytical science to problems related to formulation development in the pharmaceutical industry.

Expected graduation: December 2024

Research background:

Lyophilization (freeze-drying) has been a critical technology for the preservation, and subsequent distribution and safe administration, of innumerable protein-based drugs. While lyophilization is very effective at enhancing the long-term stability of proteins, degradation is often still inevitable. Whether it be chemical or physical, the cause of protein degradation in the lyophilized state is excipient motion¹. If the reactive species can make it to a reactive site on the protein or if the excipient can give way for the protein to unfold or aggregate, the protein will degrade; these can only occur if the excipient moves to allow for these phenomena. The objective of the present work is to develop instruments that leverage this knowledge of the excipient dynamics-protein degradation relationship to rapidly screen excipients for lyophilized protein formulations.

Techniques to directly investigate protein degradation in the solid state are few and far between. Currently, the state of the art is neutron scattering which was used in the publication that first discovered the relationship between dynamics and degradation¹. As neutron scattering is generally inaccessible, long-term stability studies usually involve long-term storage of the protein in several excipient mixtures and measurement of the degradation of the protein at several time points after the system has been reconstituted. To bridge the gap between inaccessible neutron scattering and the current need to rehydrate the protein, this work explores optical (laser-based) techniques to measure excipient dynamics in the lyophilized state.

Two promising instruments have been developed for this task. First is low-frequency Raman spectroscopy (LFR) and the second is optical Kerr effect spectroscopy (OKE). The principle for both is somewhat similar: excipients molecules move by “hopping” over one another in the solid state. These hops are low energy and due to the viscosity of the lyophilized solid, happen relatively infrequently. With LFR, I observe the number of hops that occur spontaneously and with OKE, I observe how long it takes for hops to occur by forcing the molecules to hop. The trend we see is that the less the excipients hop, the more stable the protein is.

A unique challenge of this project has been navigating spectroscopy instrument building through opaque media. In optical set-ups, it is often difficult to work with a sample that causes light to be scattered and doesn’t transmit through the sample well or at all. Likely, as a result of this and the low efficiency of Raman signal, LFR has never been used for this type of application. On the other hand, OKE has never been possible before through opaque media, but with a clever detection scheme developed by the group (patent pending), I am uniquely able to perform this study.

With these optical techniques, it is very promising that formulation candidates for lyophilized proteins can be identified very quickly, saving many resources, and potentially allowing for drug distribution to areas without cold-chain capabilities. While laser physics is not a conventional part of the chemical engineering curriculum, it has been a rewarding challenge to be at the forefront of formulation science.

One distinctive feature of my PhD journey has been how multi-faceted my project has allowed me to be in my learning and experience. While a lot of my work has been focused on instrument development, I have also worked on theory related to liquid and glass dynamics more fundamentally. With this work, related to my talk in the “Engineering Science and Fundamentals Session”, I’ve worked on developing a predictive model for liquid behavior like those that have existed for solids and gases for quite some time. Excitations, which are density fluctuations that allow for the aforementioned hops, follow a Bose-Einstein relation and can be predicted from macroscopic thermodynamic parameters such as enthalpy and entropy of melting. With the ability to predict excitations, we have seen success in relaxation time predictions. The implications of this work are that, from hops, which are the most fundamental motions that liquids and glasses can undergo, macroscopic transport processes, such as diffusion and viscosity changes, can be predicted.

On the other end of the spectrum from theory and development is applications. While exploring the behavior of my lyophilized systems, I found a second melting transition of horseradish peroxidase (HRP), an enzyme commonly used in biotechnology applications, which is not well-studied. In this work, I have explored the reversibility of HRP degradation. To this end, the team of undergraduate students that I mentor and I have found that HRP has a stable intermediate with activity that is distinct from the native state.

1. Cicerone et al., Soft Matter, 8 (2012)

Related Oral Presentations

Accounting for Excitations Allows for Liquid Phase Dynamics Predictions, 686575

Fundamental Motions: The Key to Accelerated Lyophilized Formulation Development, 686538