(6aa) Stability of Recombinant Protein-Based Bio-Pharmaceuticals: Stability in the Glassy Lyophilized State, at Various Interfaces and in Bulk Bio-Manufacturing Flows

“Stability of Protein-Based Bio-Pharmaceuticals: Stability in the Glassy Lyophilized State, At Various Interfaces and in Bulk Bio-Manufacturing Flows”

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 former 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 is 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 describing nature, yes, by all means seek simplicity, but, with respect for complexity, don’t forget to mistrust it.”

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 10 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 and to secure funding from federal agencies. 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 develop and teach new 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 co-chair of the first Gordon Research Conference GRC on Development of Bio-therapeutics and Vaccines with Prof. Christopher Roberts (Univ. of Delaware). The PI was the lead author of the successful team-proposal, whose funding led to the creation of this new GRC; website is available below.

https://www.grc.org/biotherapeutics-and-vaccines-development-conference/2019/

The 21^st 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 (bio-pharmaceuticals): large molecule drugs expressed from cells, microbes etc., for use as medicines (therapeutics) and vaccines (prophylactics).

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 and other instability pathways during these unit operations pose serious challenges to biologics development and bio-pharmaceutical manufacturing.

1) Protein Stability in Flow Fields and at Interfaces: The proposed research theme is distinct from quiescent protein aggregation (e.g. during storage), which has been studied 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 macroscopic/continuum domains. 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 air/solution and solid/solution interfaces during flow leads to formation of microscopic aggregates (~ 10 nm – ~ 1 mm), followed by larger microscopic “sub-visible particles”, as the FDA refers to them, (~1 – ~ 100 mm) and finally visible particles.

Impacts, Stakeholders and Funding Agencies: The formation of aggregates and particles poses a roadblock to therapeutic/vaccine development by increasing development cycle time and added costs. For ensuring 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), the Biomolecular Interaction Technology Center (Univ. of Delaware) 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.

2) The Physics of Protein-Protein Interactions in Concentrated Solutions: Research dedicated exclusively to the molecular/microscopic domain will probe the delicate interplay of hydrodynamics and thermodynamics in determining the stability of protein molecules, protein-protein interactions and viscosity/mutual diffusion/sedimentation behavior of protein solutions. Solution physical hydrodynamic and thermodynamic properties will be measured and interrogated against available literature theoretical models, which propose inter-relationships among them. Static light scattering (SLS) will be used to measure the osmotic second virial coefficient (B₂₂), dynamic light scattering (DLS) for quantifying the diffusion interaction parameter (k_D), sedimentation coefficient (k_S) measurements using Analytical Ultracentrifugation (AUC) and viscometry to determine protein intrinsic viscosity [η] and the Huggins coefficient k_H. The PI is a rheology expert, who routinely performs dilute solution viscometry to determine intrinsic viscosity and Huggins coefficient, and he is is also an experienced practitioner of SLS and DLS.

Impacts, Stakeholders and Funding Agencies: While theoretical predictions from colloidal models, generalized Stokes-Einstein 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 with 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 have useful consequences for 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, and this work will bridge the gap from dilute to concentrated solutions, so no extrapolations are required in understanding the thermodynamic and hydrodynamic contributions to the viscosity of concentrated protein solutions. 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.

3) Rheology and Non-Newtonian Fluid Dynamics of Protein Solutions: Inter-Relationship with Protein Stability in the Glassy State (near the glass transition temperature T_g’) and FLow Instabilities above T_g’: Since protein molecules are often prone to aggregation and are hence unstable in concentrated solutions, their development as bio-therapeutics becomes challenging. A typical short-term fix for Phase I clinical trials, where it is urgent to get the molecule into the clinic for First Time in Human (FTIH) trials to generate human tolerability and safety profiles, is to lyophilize (freeze dry) the protein formulation. Lyophilization requires the development of a formulation with appropriate bulking agents and cryo-stabilizing preservatives, and development of a freeze-drying cycle. After freezing, nucleation and primary/secondary drying. the protein and other excipients are left in the form of an elegant cake inside the glass vial. The cake is reconstituted by addition of specified quantity of sterile Water for Injection in the clinic. While the molecule is in Phase I trials in the freeze-dried solid dosage form, a stable liquid dosage form formulation is developed in the interim for Phase II clinical trials.

While lyophilization removes water, and enables rapid translation of the molecule into the clinic for FTIH milestone, trace quantities of moisture act as a plasticizer that reduces the T_g’ of the lyophilized formulation. These trace quantities of water fasten the β-relaxations (secondary relaxations associated with side chains of protein molecules), which are related to the shelf-life of the lyophilized formulation, thanks to the pioneering work of Marcus Cicerone et al. at NIST. While Cicerone et al. have used elegant inelastic neutron scattering techniques to quantify the beta-relaxations, most academic and industrial researchers cannot easily access beam lines that are only available on the beam line at a cold neutron reactor source. The PI proposes to use high frequency shear rheology and possibly also dielectric/fluorescence correlation spectroscopy to quantify the temperature and frequency dependence of these β-relaxations, as is done for polymeric glasses. High frequencies can be easily accessed with oscillatory shear rheology, and making these measurements of the β-relaxations and α-relaxation (glass transition) over a broad temperature range will enable us to determine the temperature dependence of their relaxation timescales. The temperature dependence of this a-relaxation timescale will also help elucidate the storage shelf life of protein therapeutics. Typically, these liquid formulation therapeutics are stored in cold chain at 5 ^oC, while accelerated stability tests are typically performed at 40 ^oC, to kinetically accelerate the instability pathway, which is most commonly aggregation. The PI and his team will probe universality in the temperature dependence of the a-relaxation, which is a reasonable expectation from proteins, since they form fragile glasses.

Finally, the behavior of therapeutic protein solutions as they flow out filling nozzles will be visualized using high speed microscopy to observe the different regimes that are associated with non-Newtonian fluids: dripping, jetting, etc. The delicate balance between a plethora of physical forces, such as surface tension, gravity, inertia, elasticity etc. demarcates these various regimes observed in high speed video microscopy. This investigation has not been done in the context of concentrated protein solutions of pharmaceutical relevance. This research will be conducted on a fundamental basis, and explained in terms of the flow field and protein solution rheology and physical characteristics. This work has tremendous technological importance too, as improper filling before lyophilization can lead to splashing etc., which leads to rejection of costly drug product during mandatory regulatory visual inspection. Consequently, bio-pharma companies suffer yield hits, and sometimes entire batches are rejected.

Impacts, Stakeholders and Funding Agencies: The PI will try to fundamentally study the physics of protein glasses, and translate the learning to interested bio-pharma companies that are prospective collaborators. The learning from this research will be disseminated in journals such as Molecular Pharmaceutics, Pharmaceutical Research etc. The agencies that will be approached for funding the glass transition work include the NIH, the Burroughs-Wellcome Fund and the Bill and Melinda Gates Foundation, because this work can also be translated to improve the stability of vaccines in developing countries and the development of room temperature stable vaccines by addition of excipients that raise the T_g’ above room temperature, thus kinetically retarding the degradation pathway. The funding agencies for flow visualization of protein solutions include the NSF-CBET and bio-pharmaceutical companies. That research will be published in Journal of Non-Newtonian Fluid Mechanics and Journal of Pharmaceutical Sciences.

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.

[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).

Teaching Interests:

Philosophy: I am a strong believer in teaching our undergraduates and graduate students well: they are our next generation of engineers and scientists, and deserve the same investment in teaching that my professors made in me. My pedagogical philosophy is outlined in detail below. I served as a TA for 6 semesters, and enjoy teaching.

For undergraduates, my main objective is to teach them to grasp the essential concepts of Chemical Engineering in ways that they can readily apply in their careers. My emphasis will be on teaching them how to make sense of the output from their calculations, done using whatever software package/code, and determine whether it is physically sensible and correct. It is great of they can learn how to write code. They will prove their mettle as chemical engineers if they can make sense of the inputs to and outputs of their code.

I will tailor elective courses in such a way to advanced undergraduates so that those students who have an aptitude for graduate school will be exposed to reading the literature and the process of building critical thinking skills. Having worked in the bio-pharmaceutical industry, I will build a program that prepares students for careers in this vibrant industry, which is predicted to be a key growth sector in the US in this century. Revenue from bio-pharmaceuticals (biologics) already far outstrips the revenue from small molecule drugs. I propose to develop a new-course in bio-pharma manufacturing, wherein I will teach them the basics of upstream cell culture, downstream purification and formulation/aseptic fill-finish in a cGMP (current Good Manufacturing practices) environment.

In addition to teaching them engineering, I will also start a new course for graduating seniors to teach them skills that today’s employers expect: communication (technical/regulatory writing and presentations), time management and prioritization, working in a matrixed environment, interviewing skills, workplace etiquette and communication, and soft skills (e.g. body language awareness and control): these are all critical for landing job offers after interviews and for successful career development and progression.

For graduate students, my approach is radically different. With them, my over-riding emphasis will be on development of critical thinking skills and learning how to think fast on their feet to respond effectively and sensibly to tough questions. In addition to lectures, I will hand out papers and ask them to find logical flaws in them after reading them carefully. I will also require in-class presentations, and I will develop their ability to think quickly on their feet and handle challenging questions from the audience effectively. Students will also be required to learn how to decipher the physics underlying mathematical equations. Moreover, I will work with faculty to train the graduate students in this critical skill during graduate student colloquia. I will work with faculty to encourage graduate students to spend at least one summer in industry as an intern. Most graduate students pursue careers in industry, and need hands-on early preparation for this transition, so that the industry environment is not a mystery to them when they start their careers,

The list of courses I am interested in teaching is below.

• Undergraduates
• Mass and Energy Balances

• Transport Phenomena (Momentum, Mass and Heat Transfer)

• Transition to the Workplace for ChEs (seniors only – new course to be developed)

• Process Control

• Graduate Students[1]
• Core - Fluid Mechanics; Thermodynamics/Statistical Mechanics
• Elective - Bio-pharmaceutical Manufacturing (new course – to be developed)
• Elective - Protein Solution Biophysics (new course – to be developed)
• Elective - Rheology of Complex Fluids (new course – to be developed)

[1] These courses will also be open to advanced undergrads who can satisfy appropriate prerequisites.