Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Minimal purification method enables developability assessment of recombinant proteins.

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii.

Comparison of Protein Particle Formation in IgG1 mAbs Formulated with PS20 Vs. PS80 When Subjected to Interfacial Dilatational Stress.

Polysorbates (PS) are nonionic surfactants that are commonly included in protein formulations to mitigate the formation of interfacial stress-induced protein particles and thus increase their long-term storage stability. Nonetheless, factors that dictate the efficiency of different polysorbates in mitigating protein particle formation, especially during the application of interfacial stresses, are often ill defined. Here, we used a Langmuir trough to determine the surface activity of two IgG1 monoclonal antibodies formulated with two different polysorbates (PS20 and PS80) when subjected to interfacial dilatational stress. Interfacial properties of these formulations were then correlated with characterization of subvisible protein particles measured by micro-flow imaging (MFI). Both mAbs, when formulated in PS20, demonstrate faster adsorption kinetics and higher surface activity compared to PS80 or surfactant-free formulations. Compression/expansion results suggest that when exposed to interfacial dilatational stresses, both mAb/PS20 formulations display interfacial properties of PS20 alone. In contrast, interfacial properties of both mAb/PS80 formulations suggest mAbs and PS80 are co-adsorbed to the air-water interface. Further, MFI analysis of the interface and the bulk solution confirms that PS20 is more effective than PS80 at mitigating the formation of larger particles in the bulk solution in both mAbs. Concomitantly, the efficiency of PS to prevent interface-induced protein particle formation also depended on the protein's inherent tendency to aggregate at a surfactant-free interface. Together, the studies presented here highlight the importance of determining the interfacial properties of mAbs, surfactants, and their combinations to make informed formulation decisions about the choice of surfactant.

Modeling the long-term 2-8 °C stability profiles of a live, rotavirus vaccine candidate (RV3-BB) in various liquid formulations via extrapolations of real-time and accelerated stability data.

To accelerate the formulation development of live-virus vaccine (LVV) candidates, more rapid approaches to rank-order formulations and estimate their real-time storage stability losses are needed. In this case-study, we utilize new and previously described stability data of a live, rotavirus vaccine candidate (RV3-BB) in three different liquid formulations to model and compare predicted vs. experimental RV3-BB stability profiles. Linear-regression extrapolations of limited real-time (2-8 °C) stability data and Arrhenius modeling of accelerated (15, 25, 37 °C) stability data provided predictions of RV3-BB real-time stability profiles (2-8 °C, 24 months). Good correlations of modeled versus experimental stability data to rank-order the RV3-BB formulations were achieved by employing (1) a high-throughput RT-qPCR assay to measure viral titers, (2) additional assay replicates and stability time-points, and (3) a -80 °C control for each formulation to benchmark results at each stability time-point and temperature. Instead of accumulating two-year, 2-8 °C storage stability data, the same rank-ordering of the three RV3-BB formulations could have been achieved by modeling 37°, 25°, 15° (and 2-8 °C) stability data over 1, 3 and 12 months, respectively. The results of this case-study are discussed in the context of accelerating LVV formulation development by expeditiously identifying stable formulations, estimating their shelf-lives, and determining vaccine vial monitoring (VVM) designations.

Crystallization of a nonreplicating rotavirus vaccine candidate.

Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first-principles models and preliminary experimental data. The first-principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging-drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled-up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus.

BACKGROUND: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. RESULTS: We describe a holistic approach for the molecular design of recombinant protein antigens-considering both their manufacturability and antigenicity-informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. CONCLUSIONS: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

The Pfs230 N-terminal fragment, Pfs230D1+: expression and characterization of a potential malaria transmission-blocking vaccine candidate.

BACKGROUND: Control and elimination of malaria can be accelerated by transmission-blocking interventions such as vaccines. A surface antigen of Plasmodium falciparum gametocytes, Pfs230, is a leading vaccine target antigen, and has recently progressed to experimental clinical trials. To support vaccine product development, an N-terminal Pfs230 antigen was designed to increase yield, as well as to improve antigen quality, integrity, and homogeneity. METHODS: A scalable baculovirus expression system was used to express the Pfs230D1+ construct (aa 552-731), which was subsequently purified and analysed. Pfs230D1+ was designed to avoid glycosylation and protease digestion, thereby potentially increasing homogeneity and stability. The resulting Pfs230D1+ protein was compared to a previous iteration of the Pfs230 N-terminal domain, Pfs230C1 (aa 443-731), through physiochemical characterization and in vivo analysis. The induction of functional antibody responses was confirmed via the standard membrane feeding assay (SMFA). RESULTS: Pfs230D1+ was produced and purified to an overall yield of 23 mg/L culture supernatant, a twofold yield increase over Pfs230C1. The Pfs230D1+ protein migrated as a single band via SDS-PAGE and was detected by anti-Pfs230C1 monoclonal antibodies. Evaluation by SDS-PAGE, chromatography (size-exclusion and reversed phase) and capillary isoelectric focusing demonstrated the molecule had improved homogeneity in terms of size, conformation, and charge. Intact mass spectrometry confirmed its molecular weight and that it was free of glycosylation, a key difference to the prior Pfs230C1 protein. The correct formation of the two intramolecular disulfide bonds was initially inferred by binding of a conformation specific monoclonal antibody and directly confirmed by LC/MS and peptide mapping. When injected into mice the Pfs230D1+ protein elicited antibodies that demonstrated transmission-reducing activity, via SMFA, comparable to Pfs230C1. CONCLUSION: By elimination of an O-glycosylation site, a potential N-glycosylation site, and two proteolytic cleavage sites, an improved N-terminal Pfs230 fragment was produced, termed D1+, which is non-glycosylated, homogeneous, and biologically active. An intact protein at higher yield than that previously observed for the Pfs230C1 fragment was achieved. The results indicate that Pfs230D1+ protein produced in the baculovirus expression system is an attractive antigen for transmission-blocking vaccine development.

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody.

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-CH2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

In this report we investigated, within a group of closely related single domain camelid antibodies (VH Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like VH Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.

Empirical Correction for Differences in Chemical Exchange Rates in Hydrogen Exchange-Mass Spectrometry Measurements.

A barrier to the use of hydrogen exchange-mass spectrometry (HX-MS) in many contexts, especially analytical characterization of various protein therapeutic candidates, is that differences in temperature, pH, ionic strength, buffering agent, or other additives can alter chemical exchange rates, making HX data gathered under differing solution conditions difficult to compare. Here, we present data demonstrating that HX chemical exchange rates can be substantially altered not only by the well-established variables of temperature and pH but also by additives including arginine, guanidine, methionine, and thiocyanate. To compensate for these additive effects, we have developed an empirical method to correct the hydrogen-exchange data for these differences. First, differences in chemical exchange rates are measured by use of an unstructured reporter peptide, YPI. An empirical chemical exchange correction factor, determined by use of the HX data from the reporter peptide, is then applied to the HX measurements obtained from a protein of interest under different solution conditions. We demonstrate that the correction is experimentally sound through simulation and in a proof-of-concept experiment using unstructured peptides under slow-exchange conditions (pD 4.5 at ambient temperature). To illustrate its utility, we applied the correction to HX-MS excipient screening data collected for a pharmaceutically relevant IgG4 mAb being characterized to determine the effects of different formulations on backbone dynamics.

Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1.

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies.

Instability of vaccines often emerges as a key challenge during clinical development (lab to clinic) as well as commercial distribution (factory to patient). To yield stable, efficacious vaccine dosage forms for human use, successful formulation strategies must address a combination of interrelated topics including stabilization of antigens, selection of appropriate adjuvants, and development of stability-indicating analytical methods. This review covers key concepts in understanding the causes and mechanisms of vaccine instability including (1) the complex and delicate nature of antigen structures (e.g., viruses, proteins, carbohydrates, protein-carbohydrate conjugates, etc.), (2) use of adjuvants to further enhance immune responses, (3) development of physicochemical and biological assays to assess vaccine integrity and potency, and (4) stabilization strategies to protect vaccine antigens and adjuvants (and their interactions) during storage. Despite these challenges, vaccines can usually be sufficiently stabilized for use as medicines through a combination of formulation approaches combined with maintenance of an efficient cold chain (manufacturing, distribution, storage and administration). Several illustrative case studies are described regarding mechanisms of vaccine instability along with formulation approaches for stabilization within the vaccine cold chain. These include live, attenuated (measles, polio) and inactivated (influenza, polio) viral vaccines as well as recombinant protein (hepatitis B) vaccines.

A Combined LC-MS and Immunoassay Approach to Characterize Preservative-Induced Destabilization of Human Papillomavirus Virus-like Particles Adsorbed to an Aluminum-Salt Adjuvant.

During the multi-dose formulation development of recombinant vaccine candidates, protein antigens can be destabilized by antimicrobial preservatives (APs). The degradation mechanisms are often poorly understood since available analytical tools are limited due to low protein concentrations and the presence of adjuvants. In this work, we evaluate different analytical approaches to monitor the structural integrity of HPV16 VLPs adsorbed to Alhydrogel™ (AH) in the presence and absence of APs (i.e., destabilizing m-cresol, MC, or non-destabilizing chlorobutanol, CB) under accelerated conditions (pH 7.4, 50 °C). First, in vitro potency losses displayed only modest correlations with the results from two commonly used methods of protein analysis (SDS-PAGE, DSC). Next, results from two alternative analytical approaches provided a better understanding of physicochemical events occurring under these same conditions: (1) competitive ELISA immunoassays with a panel of mAbs against conformational and linear epitopes on HPV16 VLPs and (2) LC-MS peptide mapping to evaluate the accessibility/redox state of the 12 cysteine residues within each L1 protein comprising the HPV16 VLP (i.e., with 360 L1 proteins per VLP, there are 4320 Cys residues per VLP). These methods expand the limited analytical toolset currently available to characterize AH-adsorbed antigens and provide additional insights into the molecular mechanism(s) of AP-induced destabilization of vaccine antigens.

Evaluating the Compatibility of New Recombinant Protein Antigens (Trivalent NRRV) with a Mock Pentavalent Combination Vaccine Containing Whole-Cell Pertussis: Analytical and Formulation Challenges.

Introducing new recombinant protein antigens to existing pediatric combination vaccines is important in improving coverage and affordability, especially in low- and middle-income countries (LMICs). This case-study highlights the analytical and formulation challenges encountered with three recombinant non-replicating rotavirus vaccine (NRRV) antigens (t-NRRV formulated with Alhydrogel® adjuvant, AH) combined with a mock multidose formulation of a pediatric pentavalent vaccine used in LMICs. This complex formulation contained (1) vaccine antigens (i.e., whole-cell pertussis (wP), diphtheria (D), tetanus (T), Haemophilus influenza (Hib), and hepatitis B (HepB), (2) a mixture of aluminum-salt adjuvants (AH and Adju-Phos®, AP), and (3) a preservative (thimerosal, TH). Selective, stability-indicating competitive immunoassays were developed to monitor binding of specific mAbs to each antigen, except wP which required the setup of a mouse immunogenicity assay. Simple mixing led to the desorption of t-NRRV antigens from AH and increased degradation during storage. These deleterious effects were caused by specific antigens, AP, and TH. An AH-only pentavalent formulation mitigated t-NRRV antigen desorption; however, the Hib antigen displayed previously reported AH-induced instability. The same rank-ordering of t-NRRV antigen stability (P[8] > P[4] > P[6]) was observed in mock pentavalent formulations and with various preservatives. The lessons learned are discussed to enable future multidose, combination vaccine formulation development with new vaccine candidates.

Effects of salts from the Hofmeister series on the conformational stability, aggregation propensity, and local flexibility of an IgG1 monoclonal antibody.

This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAb's backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (Tm) for three observed conformational transitions within the mAb by 6-9 °C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 °C over 12 months, as monitored by size exclusion chromatography. Backbone flexibility, as measured via H/D exchange mass spectrometry, increased in two segments in the CH2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the Tm values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the VL, CH1, and CH3 domains. In contrast, 0.5 M sulfate increased Tm values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the CH1 and VL domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the CH2 domain (especially residues 241-251 in the heavy chain), the stabilizing anion sulfate did not affect these CH2 regions.

Analytical lessons learned from selected therapeutic protein drug comparability studies.

The successful implementation of process and product changes for a therapeutic protein drug, both during clinical development and after commercialization, requires a detailed evaluation of their impact on the protein's structure and biological functionality. This analysis is called a comparability exercise and includes a data driven assessment of biochemical equivalence and biological characterization using a cadre of analytical methodologies. This review focuses on describing analytical results and lessons learned from selected published therapeutic protein comparability case studies both for bulk drug substance and final drug product. An overview of the currently available analytical methodologies typically used is presented as well as a discussion of new emerging analytical techniques. The potential utility of several novel analytical approaches to comparability studies is discussed including distribution and stability of protein drugs in vivo, and enhanced evaluation of higher-order protein structure in actual formulations using hydrogen/deuterium exchange mass spectrometry, two-dimensional nuclear magnetic resonance fingerprinting or empirical phase diagrams. In addition, new methods for detecting and characterizing protein aggregates and particles are presented as these degradants are of current industry-wide concern. The critical role that analytical methodologies play in elucidating the structure-function relationships for therapeutic protein products during the overall assessment of comparability is discussed.

The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case.

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles.

Formulation Studies to Develop Low-Cost, Orally-Delivered Secretory IgA Monoclonal Antibodies for Passive Immunization Against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a common cause for diarrheal infections in children in low- and middle-income countries (LMICs). To date, no ETEC vaccine candidates have been approved. Passive immunization with low-cost, oral formulations of secretory IgA (sIgA) against ETEC is an alternative approach to protect high-risk populations in LMICs. Using a model sIgA monoclonal antibody (anti-LT sIgA2-mAb), the stability profiles of different formulations were assessed during storage and in in vitro digestion models (mimicking in vivo oral delivery). First, by employing various physicochemical techniques and a LT-antigen binding assay, three formulations with varying acid-neutralizing capacity (ANC) were evaluated to stabilize sIgA2-mAb during stress studies (freeze-thaw, agitation, elevated temperature) and during exposure to gastric phase digestion. Next, a low-volume, in vitro intestinal digestion model was developed to screen various additives to stabilize sIgA2-mAb in the intestinal phase. Finally, combinations of high ANC buffers and decoy proteins were assessed to collectively protect sIgA2-mAb during in vitro sequential (stomach to intestine) digestion. Based on the results, we demonstrate the feasibility of low-cost, 'single-vial', liquid formulations of sIgA-mAbs delivered orally after infant feeding for passive immunization, and we suggest future work based on a combination of in vitro and in vivo stability considerations.

Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine candidate.

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.

Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 spike ferritin nanoparticle vaccine antigen produced from two different cell lines.

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.