The upper limit of protein thermostability

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Applied Biological Sciences, 1989.Includes bibliographical references (leaves 143-158).by David B. Volkin.Ph.D

Application of analytical characterization tools in process and formulation development of low cost vaccines using the ULTRA manufacturing platform

In 2016, an estimated 19.5 million infants worldwide did not receive routine life-saving vaccinations according to the World Health Organization (WHO)1. Two major limitations in improving global vaccination coverage include the costs associated with vaccine manufacturing and the challenges associated with maintaining a consistent supply. The aim of the ‘ULTRA’ project (Ultra Low-cost TRansferable Automated Platform for Vaccine Manufacturing) is to standardize the development and production of new protein subunit vaccine candidates at globally affordable costs by creating a generic, low-cost, integrated, and automated vaccine manufacturing platform. In a collaborative effort between MIT, UCL, and KU (Figure 1), state-of-the-art analytical tools will be utilized to provide well-characterized vaccine bulk and drug product facilitating process changes and reduced QC costs. Please click Additional Files below to see the full abstract

Multidimensional Methods for the Formulation of Bipharmaceuticals and Vaccines

Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA and macromolecular complexes such as viruses, virus-like particles and adjuvanted antigens is often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods, since each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecule's physical state as a function of temperature, solvent conditions, and other stress variables. We present a tutorial on the mathematical methodology, an overview of some of the experimental methods typically used, and examples of some of the previous major formulation applications. We also explore novel applications of EPDs including potential new mathematical approaches as well as possible new biopharmaceutical applications such as analytical comparability, chemical stability, and protein dynamics

Application of radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an IgG1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of Microflow Digital Imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size exclusion chromatography (SEC), Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques

Formulation development of a recombinant protein based non-replicating rotavirus (NRRV) vaccine candidate: Antigen-adjuvant-preservative interactions

Rotavirus is the leading cause of acute diarrhea and gastroenteritis among infants and young children worldwide. Over 215,000 children under five years of age die from rotavirus infection each year, mostly in developing world1. Currently two live attenuated oral rotavirus vaccines are available globally (Rotarix® and RotaTeq®) to reduce the burden of this disease with an efficacy of \u3e90% in developed countries2. Vaccine efficacy is lower, however, in developing countries due to a variety of factors3. To this end, a non-replicating rotavirus (NRRV) vaccine candidate, containing three recombinant protein antigens (P2-VP8-P[4], P2-VP8-P[6] and P2-VP8-P[8]), is being developed by PATH and its partners as a trivalent vaccine for use in the developing world4. This trivalent rotavirus vaccine candidate includes the three antigens from the most prevalent serotypes associated with \u3e90% of rotavirus gastroenteritis worldwide. In the present study, the following formulation development issues were examined: (1) establish stability-indicating physicochemical assays for a NRRV protein antigen (P[8]) bound to an aluminum hydroxide adjuvant (Alhydrogel®), which include primary and higher-order structures, chemical and conformational stability of the protein on Alhydrogel, and the ability to desorb the antigen from Alhydrogel; (2) examine the adsorptive capacity and coefficients of Alhydrogel® for the P[8] antigen in several candidate drug product formulations; (3) investigate the effects of binding to Alhydrogel® and the addition of two antimicrobial preservatives (2-phenoxyethanol or thimerosal) on the structural integrity and conformational stability of P[8], the latter of which were found to be potent destabilizers of the antigen; and (4) monitor the real-time and accelerated storage stability over 3 months of P[8] bound to Alhydrogel® in several candidate formulations with and without thimerosal at different temperatures. In the absence of preservative, the P[8] protein antigen was overall stable with only a small amount of Asn deamidation observed in samples stored under real-time (4˚C) or accelerated (25˚C) temperatures. Similarly, little (if any) changes were observed in the real-time stability of the antigen on Alhydrogel® in the presence of thimerosal. Under accelerated storage temperatures (25 or 37˚C) however, the preservative caused an increase in inter-molecular disulfide bonding, decrease of apparent enthalpy as measured by DSC, and a decrease in in-vitro antigenicity. Similar stability studies are currently ongoing with the P[4] and P[6] protein antigens. Acknowledgements: Funding provided by the Bill & Melinda Gates Foundation References: 1. Tate et al 2016. Clinical Infectious Diseases 62:S96-S105 2. Tissera et al. 2017. Human Vaccines & Immunotherapeutics 13(4):921-927 3. Glass et al. 2014. Journal of Infection 68: S9-S18. 4. Groome et al. 2017. Lancet Infectious Diseases17(8): 843-853

Two Decades of Publishing Excellence in Pharmaceutical Biotechnology

Recombinant biological products have revolutionized modern medicine by providing both remarkably effective vaccines to prevent disease and therapeutic drugs to treat a wide variety of unmet medical needs. Since the early 1980s, dozens of new therapeutic protein drugs and macromolecular vaccines have been commercialized, which have benefitted millions of patients worldwide. The pharmaceutical development of these biological products presented many scientific and technical challenges, some of which continue today with newer candidates including recombinant protein-based vaccines with novel adjuvants, peptide and RNA-based drugs, and stem cellular therapies. Compared with small molecule drugs, the characterization, stabilization, formulation, and delivery of biomolecules share common hurdles as well as unique challenges. This area of drug development research has been referred to as “pharmaceutical biotechnology”, in recognition of the critical role that recombinant DNA technology plays in the design and production of most of these biological products. Current research focus areas in this field include (i) determination of structural integrity of the primary sequence, post-translational modifications, and higher-order three dimensional shapes, (ii) assessment of physicochemical degradation pathways and their effects on biological activity and potency, (iii) formulation design and development to optimize stability and delivery, (iv) evaluating and optimizing process development steps including lyophilization and fill-finish, (v) analytical method development and applications of new instruments and data visualization tools, (vi) design and development of drug delivery approaches, and (vii) studies of biological effects including pharmacokinetics, pharmacodynamics, and adverse immunogenicity. During the early days of pharmaceutical biotechnology research, there were numerous scientific challenges because the analytical characterization approaches needed for development of recombinant biological molecules in “real world” pharmaceutical dosage forms were essentially unknown. Furthermore, understanding critical drug product manufacturing issues (e.g., stability of biological compounds during processing, storage, and shipping as well as reproducibility of fill-finish production technologies) and behavior during and after patient administration was often achieved by “on-the-job” training. Fortunately, the pioneers in the field regularly presented research at key conferences and started publishing early in pharmaceutical sciences journals such as Journal of Pharmaceutical Sciences. Recognizing this critically important new field, the then Editor of the journal, Professor Bill Higuchi, instituted a new “pharmaceutical biotechnology” category for research papers. This insightful move was coupled with an equally wise decision to recruit Dr. C. Russell Middaugh as the new Associate Editor for the new research category. As will be detailed below, under Dr. Middaugh’s diligent and expert guidance, pharmaceutical biotechnology papers have grown in number, scope, and impact over the past 20 years, and these days, the Journal of Pharmaceutical Sciences is viewed by scientific leaders in the field as the “go to” place for publication of the most important results and descriptions of innovations in pharmaceutical biotechnology

Formulation development of a stable, orally delivered live human neonatal rotavirus(rv3-bb) vaccine candidate

Rotavirus is the most common cause of gastroenteritis among children under 5 years of age leading to ~200,000 deaths in 2013.1 Rotavirus-attributed mortality can be significantly reduced by promoting global implementation of rotavirus vaccination by vaccine dosage cost reduction and optimizing vaccine efficacy in low-resource countries. Furthermore, a rotavirus vaccine administered at birth could prevent neonatal mortality and reduce the risk of intussusception 2. An oral human neonatal rotavirus vaccine candidate (RV3-BB) has been developed from the human neonatal rotavirus strain RV3 (G3P[6]) 2 , and a recently published Phase IIb clinical trial showed RV3-BB was efficacious in preventing severe rotavirus gastroenteritis via a neonatal or infant schedule in Indonesia2. The overall goals of this project are to develop and implement commercially viable bulk and drug product manufacturing processes of a stable liquid formulation for oral delivery (without pre-neutralization) that is affordable in the developing world (Fig. 1). The consortium working on this program is sponsored by the Bill and Melinda Gates Foundation between Batavia Biosciences, Murdoch Children’s Research Institute, BioFarma, and The University of Kansas. Please click Additional Files below to see the full abstract

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional “pannings” on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development

Development of stabilizing formulations of a trivalent inactivated poliovirus vaccine in a dried state for delivery in the Nanopatch™ microprojection array

The worldwide switch to inactivated polio vaccines (IPV) is a key component of the overall strategy to achieve and maintain global polio eradication. To this end, new IPV vaccine delivery systems may enhance patient convenience and compliance. In this work, we examine NanopatchTM (a solid, polymer micro-projection array) which offers potential advantages over standard needle/syringe administration including intradermal delivery and reduced antigen doses. Using trivalent IPV (tIPV) and a purpose-built evaporative dry-down system, candidate tIPV formulations were developed to stabilize tIPV during the drying process and upon storage. Identifying conditions to minimize tIPV potency losses during rehydration and potency testing was a critical first step. Various classes and types of pharmaceutical excipients (~50 total) were then evaluated to mitigate potency losses (measured through D-antigen ELISAs for IPV1, IPV2, and IPV3) during drying and storage. Various concentrations and combinations of stabilizing additives were optimized in terms of tIPV potency retention, and two candidate tIPV formulations containing a cyclodextrin and a reducing agent (e.g., glutathione), maintained ≥80% D-antigen potency during drying and subsequent storage for 4 weeks at 4˚C, and ≥60% potency for 3 weeks at room temperature with the majority of losses occurring within the first day of storage. References: * Wan, Y., et al. (in press), Development of Stabilizing Formulations of a Trivalent Inactivated Poliovirus Vaccine in a Dried State for Delivery in the Nanopatch™ Microprojection Array. Journal of Pharmaceutical Sciences. 2018. Acknowledgements: This work was funded by The World Health Organization

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions