Cambridge Healthtech Institute’s Kent Simmons recently spoke with Dr. Mark Fisher, Professor of Biochemistry and Molecular Biology at the University of Kansas Medical School, about his upcoming Keynote presentation “Capturing, Identifying and Visualizing Preaggregate Transients Using Chaperonin-Based Biolayer Interferometry Platforms”, to be delivered in the Biophysical Analysis of Biotherapeutics meeting at the 2018 PEGS event. PEGS is scheduled for April 30 - May 4, 2018 in Boston, with the biophysical analysis program set for May 2-3.
As a veteran of the industry, what are your thoughts on how the industry has changed or matured in terms of understanding the causes of aggregate formation and mechanisms of action?
Although I am not a “veteran” of industry, the largest gray area in dealing with protein aggregation is the identification of regions that are susceptible toward transient unfolding and aggregation. Much progress has been made using newer faster isolation of protein fragments that pinpoint potential regions using hydrogen deuterium exchange measurements. This knowledge can lead toward specific engineering efforts to stabilize such regions and enhance protein stability for drug efficacy. Our use of custom designed chaperonin BLI biosensors to detect transient, rapidly fluctuating regions under native non-denaturing conditions even before large scale irreversible aggregation reactions occur (stress conditions) allows us to potentially assess aggregation reactions that may occur under native conditions. Our automated systems allow us to interrogate many mAb samples at one time.
What are the challenges in assessing the kinetic stability of aggregation-prone proteins?
As stated above, the major challenge with protein therapeutics is developing stable proteins that can still function within biological systems at physiological temperatures. Proteins must remain flexible to a degree because protein dynamics and movement defines life. In more simple terms, increased protein stability can sometimes lead to a loss in biological activity. If stabilization can occur in regions that are not biologically relevant and prevent native state aggregation, then product stability can be quite helpful in extending shelf life and drug efficacy.
Can you describe your strategy to assess ligand or solution-based stability of aggregation-prone proteins using an automated chaperonin biolayer interferometry platform?
Our strategy is based on the observation that ligand based binding events translate into changes in protein dynamics. In this realm, one can observe instances where ligand binding will decrease flexibility and increase protein kinetic stability. Our chaperoin biolayer interferometry approaches can also readily identify instances where molecular events will decrease protein kinetic stability. Our primary measurement depends on the amplification of the presence of hydrophobic aggregation prone patches on the protein surface that can be recognized by a very large promiscuous chaperonin.
There are two platforms that are available for assessing protein stability. In one instance, the newly engineered GroEL BLI biosensor is dipped into protein solutions (formulation/bioprocessing/development stages) to kinetically capture dynamic transient folds. This system is also useful for assessing time dependent folding or aggregation events in solution. This method can be used as an orthogonal method to SEC where specific pre-aggregates and micro-aggregates can be detected.
In the other configuration, the protein target is attached to the biosensor surface and the kinetic stability is assessed while avoiding aggregation. This latter automated approach depends on timed denaturant pulses where proteins that are specifically orientated on biosensor surfaces are momentarily exposed to denaturing conditions that are removed in a very precisely timed solution changes. The extent of denaturation directly correlates with the amplified signal from the chaperonin binding. If a stabilizer is present during the denaturation phase and delays the kinetic denaturation, the signal from the chaperonin binding is simply diminished. The resulting kinetically controlled denaturation isotherms are highly reproducible and overwhelming statistically significant. The beauty of this approach is that it works with any aggregation-prone protein that folds into a three-dimensional structure. Furthermore, comparison of mutant folds with native folds via the denaturation pulse isotherm method may be a valid approach toward designing small molecule stabilizers to develop a personalized medicine approach to disease.
How well does your strategy integrate with other analytical techniques to accelerate decision making in stability and bioprocessing streams?
The obvious advantage of our particular biosensor surface system is that the captured protein that partition onto the GroEL biosensors can be released using ATP treatments and released products can be identified in microvolume aliquots using sensitive MS identification protocols (release femtomole quantities for ID). In addition, we have recently found that the chaperonin biosensors can capture small aggregates that can be collected in microvolumes and visualized with low resolution negative stain electron microscopy. The ability to use microvolume quantities can accelerate product assessment, small aggregate complex formation and stability without using large volumes of material. In addition, the BLI biosensor technologies can directly assess any concentrated protein solution. Our biosensor systems can capture transient populations within bioprocess streams or within final formulation solutions.
Speaker Biography:
Mark T. Fisher, PhD, Professor, Biochemistry and Molecular Biology, University of Kansas Medical Center
Dr. Mark Fisher received his BS degree in Chemistry from Purdue in 1982 and a Ph.D. in Biochemistry from the University of Illinois in 1987 under Dr. Stephen Sligar where he examined how cytochrome P450s biophysically controlled drug clearance rates by modulating electron transfer rates. He subsequently became a Postdoctoral fellow at NIH under Dr. Earl Stadtman until 1992 where he was among the pioneers in research to understand how molecular chaperones control protein folding and serve as protein homeostasis buffers in the cell. He is currently a full Professor in the Biochemistry and Molecular Biology Department at the University of Kansas Medical Center. His current research is focused on capturing or reversing protein misfolding/unfolding intermediates using various chaperonin detection based biolayer interferometry (BLI) platforms. These platforms can be used to test in silico drug design and validation of protein stability enhancements. This approach is used to accelerate drug discovery pipelines for novel protein stabilizers. For general protein formulation analysis, the Fisher laboratory has developed chaperonin BLI biosensors to capture and detect transient preaggregate species that appear prior to large scale aggregation. In some instances, these initial captured species can be released into micro volume quantities that can be visualized by electron microscopy and identified by highly sensitive Mass spectroscopy methods. In some instances, the 3D reconstruction of captured protein species is accomplished through specific biosensor release of captured proteins into micro volume aliquots followed by single particle reconstruction analysis. In all cases, the overreaching goal of this biotechnology approach is to detect, identify and, in some cases, visually pinpoint defects in protein sample integrity (aggregation prone regions) using high throughput approaches.