(7jf) Fundamental Molecular Biophysics, Rheology and Thermodynamics to Elucidate Protein Stability in Flow Fields and Protein-Protein Interactions in Concentrated Solutions

Principal Investigator: Dr. Jai A. Pathak, Vaccine Production Program, Vaccine Research
Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health
“Fundamental Molecular Biophysics, Rheology and Thermodynamics to Elucidate Protein Stability in Flow Fields and Protein-Protein Interactions in Concentrated Solutions”

The 21st century poses global challenges to mankind, especially in harnessing alternate sources of clean energy, responding to climate change, ensuring abundant food and water supply and revolution healthcare/medicine. In healthcare/medicine, bio-technology has unleashed a revolution since the discovery of recombinant DNA technology, which has unleashed the discovery, development and commercialization/regulatory approval of numerous biologics: large molecule drugs expressed from cell, microbes etc., for use as medicines (therapeutics) and vaccines (prophylactics).

Research Interests:
Chemical engineers have made prolific contributions in bio-technology to the science of upstream technology (protein expression) and downstream technology (protein purification), yet, numerous scientific/technical challenges abound. Protein stability towards aggregation and fragmentation degradation pathways during flow encountered ubiquitously in bio-manufacturing: flow during cell culture in bio-reactors, flow in chromatography columns for purification, flow in formulation vessels and filling piston/peristaltic/time- pressure pumps and fill nozzles during aseptic (sterile) fill/finish of drug products, flow during shipment of finished liquid drug product in vials/syringes and flow in syringes and other novel devices for self- administration or clinical administration. It is impossible to expresses, purify, process, ship and deliver protein drug products to patients without subjecting them to various flow fields: shear flow, extensional flow etc., wherein protein molecules in concentrated solution encounter a wide range of shear and extensional rates. Protein aggregation during these steps poses serious challenges to biologics development. The proposed research theme is distinct from quiescent protein aggregation (e.g. during storage), which has been studied with unique originality and extensively by Prof. Christopher Roberts (Univ. of Delaware) and his research group; the work proposed by the PI aims to study the interplay of thermodynamics, flow fields, protein solution rheology and surfaces/interfaces on protein stability during flow. The highly non- Newtonian rheology of concentrated protein solutions stems from hydrodynamic and thermodynamic interactions, and is also determined by the presence of solid/solution and air/solution interfaces. The effects of flow fields on thermodynamic phase behavior, which are well documented in synthetic polymer solutions, have not been scrutinized for protein solutions. This fundamental phenomenon has practical consequences
for bio-manufacturing,

This research will bridge the molecular, mesoscopic and m a croscopic/continuum dom a ins. Bulk rheology (macro and micro) and interfacial rheology (interfacial shear and dilation) will be applied in conjunction with scattering (light and neutrons), in-situ circular dichroism, intrinsic/extrinsic fluorescence, optical microscopy (flow visualization) etc. to pinpoint how the unfolding of protein molecules during flow leads to formation of microscopic aggregates (~ 10 nm – ~ 1 µm), and larger microscopic sub-visible particles (~1 – ~ 100 µm) and finally larger visible particles. The formation of aggregates and particles poses a roadblock to therapeutic/vaccine development by increasing development cycle time and added costs. For
providing patient safety against adverse immunogenic reactions, regulatory agencies such as the FDA, EMEA
etc. have strict regulations for the maximum aggregate content and proteinaceous particle size/count that can be injected into patients. Statutory compliance with these regulatory requirements ensures that the bio- pharmaceutical industry will be a key stakeholder in this basic research, as development scientists do not have the time, resources and expertise to tackle these challenges at a fundamental scientific level; strict
development timeline requirements only allow for engineering quick-fix solutions. This work will proceed on
two parallel tracks: with biophysically well-characterized model proteins for proof of concept development and rapid publication, and with proprietary proteins in collaboration with bio-pharma companies. A
collaboration with protein expression and purification experts in academia will be initiated to leverage the upstream/downstream expertise required for supply of proteins, antibodies etc. The National Institute for Innovation in Biopharmaceuticals (NIIMBL), a public-private consortium dedicated to advancing Biopharmaceutical Manufacturing Innovation (NIST/US Dept. of Commerce), and major bio-pharmaceutical companies will be approached for funding. This research will be disseminated in the following journals:

Journal of Pharmaceutical Sciences, Biotechnology Bioengineering, Journal of Rheology, Soft Matter etc.

Research dedicated exclusively to the m olecula r/m icroscopic dom ain will probe the delicate interplay of hydrodynamics and thermodynamics in determining the stability of protein molecules, protein-protein interactions and viscosity of protein solutions. Physical properties will be measured and interrogated against available colloidal theories, with adequate accounting for the contributions of the macromolecular nature of proteins. Static light scattering will be used to measure the osmotic second virial coefficient, dynamic light scattering for quantifying the diffusion interaction parameter, sedimentation measurements using Analytical Ultracentrifugation (AUC) and viscometry to determine protein intrinsic viscosity and the Huggins coefficient. While theoretical predictions from colloidal models and mode coupling theory are available for the inter-relationship amongst these hydrodynamic and thermodynamic quantities, they have not been interrogated in sufficient depth to elucidate the correct physics. This work will be done strictly in the
hydrodynamic/rheology and thermodynamics realm, with multi-domain Immunoglobulins and other globular
proteins in the public domain. The funding agencies for this work will be NIH - NIBIB (primary) and NSF - CBET (secondary). While this investigation will be at a fundamental level, its significance and impacts will be translated and made available to industry formulations scientists who perform these measurements routinely to determine protein-protein interactions, stability and high solution viscosity during the development of high concentration liquid protein formulations. Biophysical and thermodynamic measurements are done in dilute solution conditions, while HCLFs are far from dilute: inter-molecular hydrodynamic and thermodynamic interactions must be accounted for; this work will bridge the gap from dilute to concentrated solutions, so no extrapolations are required. A wealth of knowledge can be extracted by performing these measurements on purified well-characterized proteins. The learning from this research will be disseminated in journals like Biophysical Journal, Journal of Chemical Physics, Journal of Physical Chemistry, Physical Review Letters, etc.
The PI and his team will present at AIChE Annual Meeting, ACS- American Chemical Society Biotech Meeting., Biophysical Society Annual Meeting, American Physical Society March Meeting and the Society of Rheology Annual Meeting.
Peer Recognition, Key Strengths, Originality and Differentiation: The PI has (co)-authored 32 peer- reviewed publications in respected journals. The impact of the PI’s recent research, published with his industry post-doctoral fellow, Dr. Prasad Sarangapani, 1 at MedImmune/AstraZeneca, was recognized by Prof. John Prausnitz (Dept. of Chemical Engineering, UC Berkeley), who reviewed the paper and was invited by the editors of Biophysical Journal to write a “New & Notable” Comment. Prof Prausnitz wrote about 2 the significance and impact of the work of Sarangapani et al.:

“Many like me will be secretly unhappy about the demise of the colloidal theory of globular protein solutions. Although we knew that this theory was “sick,” we hoped that it might “recover.” But now after the report of Sarangapani et al., the colloid-like theory id dead. We can take comfort in the remark of Sarangapani et al., that while scientifically

erroneous, the colloid-like theory may nevertheless be useful for some purposes in biotechnology. While we mourn with sadness, we also owe much thanks to Sarangapani et al., that when escribing nature, yes, by all means seek simplicity, but, with respect for complexity, don’t forget to mistrust it.”

1 “Critical Examination of the Colloidal Particle Model of Globular Models” Prasad S. Sarangapani, Steven D. Hudson, Ronald L. Jones, Jack F. Douglas and Jai A. Pathak. Biophysical Journal, 108, 724 – 737 (2015).

2 “The Fallacy of Misplaced Concreteness” John Prausnitz, Biophysical Journal, 108, 453-454 (2015).

The PI is a recognized expert in the solution rheology and bio-physics of proteins: he delivered an invited
plenary lecture at the XVII International Conference of Rheology (Kyoto, Japan, Aug. 2016). He has nearly

9 years of experience in bio-physics. The PI has identified these research problems during his career, and has trained a post-doctoral fellow and graduate students while in industry. The PI has 11 publications in biophysics, and leverages his knowledge of macromolecular physics, fluid mechanics and rheology
extensively. The PI has an extensive network of domestic and international collaborators in academia, industry and in federal labs (NIST, NIH etc.), which he will rely upon to get the appropriate subject matter experts involved in the inter-disciplinary research program proposed here. This network will also be leveraged for funding. The PI can perform fundamental scientific inquiry in problems of relevance to industry, which will enable rigorous training of Chemical Engineers as Ph.D. scientists. The PI will also teach courses to ChE graduate and undergraduate students to mentor them for jobs in the bio-tech. industry, and will involve undergraduates in research, with special encouragement to minorities and female students. The PI is the lead author on a proposal with Prof. Christopher Roberts (Univ. of Delaware), Dr. Michael Tarlov (NIST) and
Dr. Linda Narhi (Amgen) to initiate a new Gordon Research Conference (GRC) in Development of Bio- therapeutics and Vaccines; the revised proposal is currently under review with the GRC board.
Teaching Interests:

1) Undergraduates
• Mass and Energy Balances
• Transport Phenomena (Momentum, Mass and Heat Transfer)
• Chemical Engineering Thermodynamics
• Process Control
2) Graduate Students3
• Core - Fluid Mechanics; Thermodynamics
• Elective - Bio-pharmaceutical Manufacturing
• Elective - Protein Solution Biophysics
• Elective - Statistical Mechanics
• Elective - Rheology of Complex Fluids

3 These courses will also be open to advanced undergrads who can satisfy appropriate prerequisites.