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Once Covid-19 vaccines become available, 5–10 billion vaccine doses should be globally distributed, stored and administered. In this commentary, we discuss how this enormous challenge could be addressed for viral vector-based Covid-19 vaccines by learning from the wealth of formulation development experience gained over the years on stability issues related to live attenuated virus vaccines and viral vector vaccines for other diseases. This experience has led –over time– to major improvements on storage temperature, shelf-life and in-use stability requirements. First, we will cover work on 'classical' live attenuated virus vaccines as well as replication competent viral vector vaccines. Subsequently, we address replication deficient viral vector vaccines. Freeze drying and storage at 2–8 °C with a shelf life of years has become the norm. In the case of pandemics with incredibly high and urgent product demands, however, the desire for rapid and convenient distribution chains combined with short end-user storage times require that liquid formulations with shelf lives of months stored at 2–8 °C be considered. In confronting this "perfect storm" of Covid-19 vaccine stability challenges, understanding the many lessons learned from decades of development and manufacturing of live virus-based vaccines is the shortest path for finding promising and rapid solutions. [ABSTRACT FROM AUTHOR]

The photodegradation of a human IgG1 monoclonal antibody has been examined in a high concentration (100 mg/mL) liquid formulation. It was observed that a yellowish color is generated when the formulation is exposed to intense and prolonged light exposure, and this discoloration occurs along with a loss in bioactivity. Extensive analytical characterization was performed to determine light induced degradation pathways that occur during exposure to intense light of ICH photodegradation conditions. It has been shown that the monoclonal antibody undergoes a combination of physical and chemical reactions under these conditions, including covalent aggregate formation, fragmentation at the hinge region, oxidation of Trp, His, and Met residues, and deamidation of Asn residues. Oxidation of Trp 94 and deamidation of Asn 93, located in the light chain CDR region, correlates with loss of bioactivity under these conditions. A series of formulation experiments were performed to elucidate the impact of the extent of light exposure, oxygen, protein concentration, and solution pH on the photostability of the formulation. Results demonstrated that photodegradation of the IgG, after intensive light exposure, can be prevented by proper secondary packaging. In addition, it is also shown that a high concentration, liquid dosage form of a human monoclonal antibody is stable upon exposure to the ambient light conditions encountered during routine manufacturing, long-term storage, and administration with proper design of formulation conditions, the primary container as well as the secondary package. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3117–3130, 2009 [ABSTRACT FROM AUTHOR]

The human papillomavirus (HPV) virus-like particles (VLPs) produced by recombinant expression systems are promising candidate vaccine antigens for prevention of cervical cancers as well as genital warts. However, expression of HPV type 6, 11, and 16 L1 proteins in Saccharomyces cerevisiae yielded irregularly shaped, broadly distributed VLPs smaller in size (30–50 nm) than expected (60 nm). In this study, we demonstrate that these HPV VLPs can be disassembled into the constituent capsomers (L1 pentamers) by incubation at low ionic strength and elevated pH in the presence of relatively low concentration of reducing agents. Following the removal of reducing agents, lowering of pH and increasing of ionic strength, the capsomers spontaneously reassembled into homogenous, 60-nm VLPs characterized by significantly enhanced structural stability and improved immunogenicity. In order to achieve quantitative recovery of HPV VLPs, the disassembly/reassembly process was further optimized by use of high ionic strength (>0.5 M sodium chloride) to prevent aggregation of VLPs. The reassembled VLPs possess an architectural structure very similar to that of the natural HPV virus particles. This development illustrates how the natural, in vivo mechanisms facilitating cell entry and virus replication can be utilized to achieve an optimal, in vitro assembly state of yeast-expressed HPV VLPs. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2195–2206, 2006 [ABSTRACT FROM AUTHOR]

As mRNA vaccines became the frontrunners in late-stage clinical trials to fight the COVID-19 pandemic, challenges surrounding their formulation and stability became readily apparent. In this commentary, we first describe company proposals, based on available public information, for the (frozen) storage of mRNA vaccine drug products across the vaccine supply chain. We then review the literature on the pharmaceutical stability of mRNA vaccine candidates, including attempts to improve their stability, analytical techniques to monitor their stability, and regulatory guidelines covering product characterization and storage stability. We conclude that systematic approaches to identify the key physicochemical degradation mechanism(s) of formulated mRNA vaccine candidates are currently lacking. Rational design of optimally stabilized mRNA vaccine formulations during storage, transport, and administration at refrigerated or ambient temperatures should thus have top priority in the pharmaceutical development community. In addition to evidence of human immunogenicity against multiple viral pathogens, including compelling efficacy results against COVID-19, another key strength of the mRNA vaccine approach is that it is readily adaptable to rapidly address future outbreaks of new emerging infectious diseases. Consequently, we should not wait for the next pandemic to address and solve the challenges associated with the stability and storage of formulated mRNA vaccines. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This work describes Part 2 of multi-dose formulation development of a Human Papillomavirus (HPV) Virus-Like Particle (VLP) based vaccine (see Part 1 in companion paper). Storage stability studies with candidate multi-dose formulations containing individual or combinations of seven different antimicrobial preservatives (APs) were performed with quadrivalent HPV VLP (6, 11, 16, 18) antigens adsorbed to aluminum-salt adjuvant (Alhydrogel®). Real-time (up to two years, 2-8°C) and accelerated (months at 25 and 40°C) stability studies identified eight lead candidates as measured by antigen stability (competitive ELISA employing conformational serotype-specific mAbs), antimicrobial effectiveness (modified European Pharmacopeia assay), total protein content (SDS-PAGE), and AP concentration (RP-UHPLC). The AH-adsorbed HPV18 VLP component was most sensitive to AP-induced destabilization. Optimal quadrivalent antigen storage stability while maintaining antimicrobial effectiveness was observed with 2-phenoxyethanol, benzyl alcohol, chlorobutanol, and 2-phenoxyethanol + benzyl alcohol combination. Interestingly, for single-AP containing multi-dose formulations, this rank-ordering of storage stability did not correlate with previously reported biophysical measurements of AP-induced antigen destabilization. Moreover, other APs (e.g., m-cresol, phenol, parabens) described by others for inclusion in multi-dose HPV VLP formulations showed suboptimal stability. These results suggest that each HPV VLP vaccine candidate (e.g., different serotypes, expression systems, processes, adjuvants) will require customized multi-dose formulation development. [ABSTRACT FROM AUTHOR]

The development of multi-dose, subunit vaccine formulations can be challenging since antimicrobial preservatives (APs) often destabilize protein antigens. In this work, we evaluated Human Papillomavirus (HPV) Virus-Like Particles (VLPs) to determine if combining different APs used in approved parenteral products, each at lower concentrations than used alone, would maintain both antimicrobial effectiveness and antigen stability. To identify promising AP combinations, two different screening strategies were utilized: (1) empirical one-factor-at-a-time (OFAT) and (2) statistical design-of-experiments (DOE). Seven different APs were employed to screen for two- and three-AP combinations using high-throughput methods for antimicrobial effectiveness (i.e., microbial growth inhibition assay and a modified European Pharmacopeia method) and antigen stability (i.e., serotype-specific mAb binding to conformational epitopes of HPV6, 11, 16 VLPs by ELISA). The OFAT and DOE approaches were complementary, such that initial OFAT results (and associated lessons learned) were subsequently employed to optimize the combinations using DOE. Additional validation experiments confirmed the final selection of top AP-combinations predicted by DOE modeling. Overall, 20 candidate multi-dose formulations containing two- or three-AP combinations were down-selected. As described in Part 2 (companion paper), long-term storage stability profiles of aluminum-adjuvanted, quadrivalent HPV VLP formulations containing these lead candidate AP combinations are compared to single APs. [ABSTRACT FROM AUTHOR]

Mechanical agitation of monoclonal antibody (mAb) solutions often leads to protein particle formation. In this study, various formulations of an immunoglobulin G (IgG) 1 mAb were subjected to different controlled interfacial stresses using a Langmuir trough, and protein particles formed at the interface and measured in bulk solution were characterized using atomic force microscopy and flow digital imaging. Results were compared to mAb solutions agitated in glass vials and unstressed controls. At lower pH, mAb solutions exhibited larger hysteresis in their surface pressure versus area isotherms and increased number of particles in bulk solution, when subjected to interfacial stresses. mAb samples subjected to 750-1000 interfacial compression-expansion cycles in 6 h contained high particle numbers in bulk solution, and displayed similar particulation trends when agitated in vials. At compression rates of 50 cycles in 6 h, however, particle levels in mAb solutions were comparable to unstressed controls, despite protein aggregates being present at the air-solution interface. These results suggest that while the air-solution interface serves as a nucleation site for initiating protein aggregation, the number of protein particles measured in bulk mAb solutions depends on the total number of compression cycles that proteins at the air-solution interface are subjected to within a fixed time. [ABSTRACT FROM AUTHOR]

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed. The four monovalent AH-adsorbed HPV VLPs were then characterized using these down-selected methods. Second, stability-indicating competitive ELISA assays were developed using HPV serotype-specific neutralizing mAbs, to monitor relative antibody binding profiles of the four AH-adsorbed VLPs during storage. Third, concentration-dependent preservative-induced destabilization of HPV16 VLPs was demonstrated by addition of eight preservatives found in parenterally administered pharmaceuticals and vaccines, as measured by ELISA, dynamic light scattering, and differential scanning calorimetry. Finally, preservative stability and effectiveness in the presence of vaccine components were evaluated using a combination of RP-UHPLC, a microbial growth inhibition assay, and a modified version of the European Pharmacopoeia assay (Ph. Eur. 5.1.3). Results are discussed in terms of analytical challenges encountered to identify and develop high-throughput methods that facilitate multi-dose formulation development of aluminum-adjuvanted protein-based vaccine candidates. [ABSTRACT FROM AUTHOR]

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing. [ABSTRACT FROM AUTHOR]

Rotavirus infections remain a leading cause of morbidity and mortality among infants residing in low- and middle-income countries. To address the large need for protection from this vaccine-preventable disease we are developing a trivalent subunit rotavirus vaccine which is currently being evaluated in a multinational Phase 3 clinical trial for prevention of serious rotavirus gastroenteritis. Currently, there are no universally accepted in vivo or in vitro models that allow for correlation of field efficacy to an immune response against serious rotavirus gastroenteritis. As a new generation of non-replicating rotavirus vaccines are developed the lack of an established model for evaluating vaccine efficacy becomes a critical issue related to how vaccine potency and stability can be assessed. Our previous publication described the development of an in vitro ELISA to quantify individual vaccine antigens adsorbed to an aluminum hydroxide adjuvant to address the gap in vaccine potency methods for this non-replicating rotavirus vaccine candidate. In the present study, we report on concordance between ELISA readouts and in vivo immunogenicity in a guinea pig model as it relates to vaccine dosing levels and sensitivity to thermal stress. We found correlation between in vitro ELISA values and neutralizing antibody responses engendered after animal immunization. Furthermore, this in vitro assay could be used to demonstrate the effect of thermal stress on vaccine potency, and such results could be correlated with physicochemical analysis of the recombinant protein antigens. This work demonstrates the suitability of the in vitro ELISA to measure vaccine potency and the correlation of these measurements to an immunologic outcome. [ABSTRACT FROM AUTHOR]

Although many subcutaneously (s.c.) delivered, high-concentration antibody formulations (HCAF) have received regulatory approval and are widely used commercially, formulation scientists are still presented with many ongoing challenges during HCAF development with new mAb and mAb-based candidates. Depending on the specific physicochemical and biological properties of a particular mAb-based molecule, such challenges vary from pharmaceutical attributes e.g., stability, viscosity, manufacturability, to clinical performance e.g., bioavailability, immunogenicity, and finally to patient experience e.g., preference for s.c. vs. intravenous delivery and/or preferred interactions with health-care professionals. This commentary focuses on one key formulation obstacle encountered during HCAF development: how to maximize the dose of the drug? We examine methodologies for increasing the protein concentration, increasing the volume delivered, or combining both approaches together. We discuss commonly encountered hurdles, i.e., physical protein instability and solution volume limitations, and we provide recommendations to formulation scientists to facilitate their development of s.c. administered HCAF with new mAb-based product candidates. [ABSTRACT FROM AUTHOR]

Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m -cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4 °C, 25 °C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.g., the parabens and chlorobutanol were least destabilizing). We used hydrogen exchange-mass spectrometry (HX-MS) to better understand the destabilizing effects of temperature and preservative on backbone flexibility of AH-adsorbed P[4]. Thimerosal addition immediately increased the backbone flexibility across much of the AH-adsorbed P[4] protein backbone (except the N-terminal P2 region and residues G17-Y 38), and further increase in P[4] backbone flexibility was observed after storage (4 °C, 4 weeks). HX-MS analysis of AH-adsorbed P[4] stored for 4 weeks at 25 °C revealed structural alterations in some regions of the epitope involved in P[4] specific mAb binding. These combined results are discussed in terms of a generalized workflow for multi-dose vaccine formulation development for recombinant protein antigens. [ABSTRACT FROM AUTHOR]

Formation of submicron and subvisible protein particles (0.1–100 μm) present a major obstacle during processing and storage of therapeutic proteins. While protein aggregation resulting in particle formation is well-understood in bulk solution, the mechanisms of aggregation due to interfacial stresses is less understood. Particularly, in this study, we focus on understanding the combined effect of temperature and application of interfacial dilatational stresses, on interface-induced protein particle formation, using two industrially relevant monoclonal antibodies (mAbs). The surface activity of Molecule C (MC) and Molecule B (MB) were measured at room temperature (RT) and 4 °C in the absence and presence of interfacial dilatation stress using a Langmuir trough. These results were correlated with Micro-flow imaging (MFI) to characterize formation of subvisible protein particles at the interface and in the bulk solution. Our results show that the surface activity for both proteins is temperature dependent. However, the extent of the impact of temperature on the mechanical properties of the monomolecular protein films when subjected to dilatational stresses is protein dependent. Protein particle analysis provided evidence that protein particles formed in bulk solution originate at the interface and are dependent on both application of thermal stresses and interfacial dilatational stresses. In the absence of any interfacial stresses, more and larger protein particles were formed at the interface at RT than at 4 °C. When mAb formulations are subjected to interfacial dilatational stresses, protein particle formation in bulk solution was found to be temperature dependent. Together our results validate that mAb solutions maintained at 4 °C can lower the surface activity of proteins and reduce their tendency to form interface-induced protein particles both in the absence and presence of interfacial dilatational stresses. [ABSTRACT FROM AUTHOR]

Protein instability and immunogenicity are two main roadblocks to the clinical success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address immunogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both conventional and novel therapeutic protein dosage forms. Next, we provide a summary of the stresses on proteins arising during preparation of drug delivery systems and subsequent in vivo release. We note the challenges and difficulties in achieving the absolute requirement of quantitatively assessing the degradation of protein molecules in a drug delivery system. We describe the potential roles for academic research in further improving protein stability and developing new analytical technologies to detect protein degradation byproducts in novel drug delivery systems. Finally, we provide recommendations for the appropriate approaches to formulation design and assay development to ensure that stable, minimally immunogenic formulations of therapeutic proteins are created. These approaches should help to increase the probability that novel drug delivery systems for sustained protein release will become more readily available as effective therapeutic agents to treat and benefit patients. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:946-954, 2012 [ABSTRACT FROM AUTHOR]

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 are 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 because 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. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4171-4197, 2011 [ABSTRACT FROM AUTHOR]

• A low-cost manufacturing process for a neonatal rotavirus vaccine was developed. • The formulation process developed resulted in a stable liquid vaccine at 2–8 °C. • No pretreatment of vaccinees with antacid needed before oral administration. • Tech. transfer package includes manufacturing, formulation, assays and COGs model. Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia. To further develop this vaccine candidate, a well-documented and low-cost manufacturing process is required. A target fully loaded cost of goods (COGs) of ≤$3.50 per course of three doses was set based on predicted market requirements. COGs modelling was leveraged to develop a process using Vero cells in cell factories reaching high titers, reducing or replacing expensive reagents and shortening process time to maximise output. Stable candidate liquid formulations were developed allowing two-year storage at 2–8 °C. In addition, the formulation potentially renders needless the pretreatment of vaccinees with antacid to ensure adequate gastric acid neutralization for routine oral vaccination. As a result, the formulation allows small volume dosing and reduction of supply chain costs. A dose ranging study is currently underway in Malawi that will inform the final clinical dose required. At a clinical dose of ≤6.3 log 10 FFU, the COGs target of ≤$3.50 per three dose course was met. At a clinical dose of 6.5 log 10 FFU, the final manufacturing process resulted in a COGs that is substantially lower than the current average market price, 2.44 USD per dose. The manufacturing and formulation processes were transferred to BioFarma in Indonesia to enable future RV3-BB vaccine production. [ABSTRACT FROM AUTHOR]

A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries. [ABSTRACT FROM AUTHOR]

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed. [ABSTRACT FROM AUTHOR]

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2–8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery. • Formulation development for oral delivery of a live, rotavirus vaccine candidate (RV3-BB) is described. • RV3-BB virus stability during in vivo oral delivery was mimicked with two different in vitro gastric digestion models. • Pre-neutralization with infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. • Formulation excipients (succinate, acetate, adipate) minimized acidic pH inactivation of RV3-BB without preneutralization. [ABSTRACT FROM AUTHOR]

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality. [ABSTRACT FROM AUTHOR]

Smallpox, a contagious and deadly disease caused by variola virus, was eradicated by a strategy that included vaccination with vaccinia virus, a live-virus vaccine. Because the threat of bioterrorism with smallpox persists and infections with zoonotic poxvirus infections like monkeypox continue, and there may be a time when an alternative vaccine platform is needed, recombinant-subunit vaccine strategies for poxviruses have been pursued. Our prior work focused on understanding the immune responses generated to vaccine-formulations containing the virus protein L1. In this work, we examine vaccine-formulations with additional key protein targets: A33 and B5 (components of the extracellular virus) and another protein on the mature virus (A27) adjuvanted with aluminum hydroxide (AH) with and without CpG- oligonucleotide. Each vaccine was formulated to allow either adsorption or non-adsorption of the protein (and CpG) to AH. Mice given a prime and single boost produced long-lasting antibody responses. A second boost (given ~5-months after the first) further increased antibody titers. Similar to our prior findings with L1 vaccine-formulations, the most protective A33 vaccine-formulations included CpG, resulted in the generation of IgG2a-antibody responses. Unlike the prior findings with L1 (where formulations that adsorbed both the protein and the CpG to AH resulted in 100% survival after challenge and minimal weight loss), the AH-adsorption status of A33 and CpG did not play as important a role, since both AH-adsorbed and non-adsorbed groups lost weight after challenge and had similar survival. Vaccination with B5-formulations gave different results. While CpG-containing formulations were the only ones that generated IgG2a-antibody responses, the vaccine-formulation that adsorbed B5 to AH (without CpG) was as equally effective in protecting mice after challenge. These results indicate that the mechanism of how antibodies against A33 and B5 protect differ. The data also show the complexity of designing optimized vaccine-formulations containing multiple adjuvants and recombinant protein-based antigens. [ABSTRACT FROM AUTHOR]

A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation. [ABSTRACT FROM AUTHOR]

Although live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments. [ABSTRACT FROM AUTHOR]

In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw–induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular β-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti–heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world. [ABSTRACT FROM AUTHOR]

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration. [ABSTRACT FROM AUTHOR]

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na 2 SO 4), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride > trimethylphenylammonium iodide > tryptophan amide hydrochloride > ethanol as measured by various biophysical techniques. Conversely, addition of Na 2 SO 4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C. [ABSTRACT FROM AUTHOR]

Live attenuated viral vaccine/vector candidates are inherently unstable and infectivity titer losses can readily occur without defining appropriate formulations, storage conditions and clinical handling practices. During initial process development of a candidate vaccine against HIV-1 using a recombinant Human Cytomegalovirus vector (rHCMV-1), large vector titer losses were observed after storage at 4 °C and after undergoing freeze-thaw. Thus, the goal of this work was to develop candidate frozen liquid formulations of rHCMV-1 with improved freeze-thaw and short-term liquid stability for potential use in early clinical trials. To this end, a virus stability screening protocol was developed including use of a rapid, in vitro cell-based immunofluorescence focus assay to quantitate viral titers. A library of ∼50 pharmaceutical excipients (from various known classes of additives) were evaluated for their effect on vector stability after freeze-thaw cycling or incubation at 4 °C for several days. Certain additives including sugars and polymers (e.g., trehalose, sucrose, sorbitol, hydrolyzed gelatin, dextran 40) as well as removal of NaCl (lower ionic strength) protected rHCMV-1 against freeze-thaw mediated losses in viral titers. Optimized solution conditions (e.g., solution pH, buffers and sugar type) slowed the rate of rHCMV-1 titer losses in the liquid state at 4 °C. After evaluating various excipient combinations, three new candidate formulations were designed and rHCMV-1 stability was benchmarked against both the currently-used and a previously reported formulation. The new candidate formulations were significantly more stable in terms of reducing rHCMV-1 titer losses after 5 freeze-thaw cycles or incubation at 4 °C for 30 days. This case study highlights the utility of semi-empirical design of frozen liquid formulations of a live viral vaccine candidate, where protection against infectivity titer losses due to freeze-thaw and short-term liquid storage are sufficient to enable more rapid initiation of early clinical trials. [ABSTRACT FROM AUTHOR]

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies. [ABSTRACT FROM AUTHOR]

Abstract In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration. [ABSTRACT FROM AUTHOR]

Abstract A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration, and zeta potential using a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a quantitative polymerase chain reaction assay for quantitation of viral genome copy number (r2 = 0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibited viral vector particle loss under these conditions included proline, lactose, and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations. [ABSTRACT FROM AUTHOR]

Abstract The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from −80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at −20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed. [ABSTRACT FROM AUTHOR]

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris , 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments. [ABSTRACT FROM AUTHOR]

The effects of 2 squalene-based emulsion adjuvant systems (MedImmune emulsion 0 [ME.0] and Stable Emulsion [SE]) on the structure and stability of the recombinant protein antigen alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection, were investigated using Fourier-transform infrared spectroscopy and both steady-state and time-resolved intrinsic fluorescence spectroscopy as well as differential scanning calorimetry (DSC). A component study, performed to identify the effects of the individual emulsion's components, showed negligible interactions between AT and ME.0. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a significant blue shift in the fluorescence spectra relative to that of AT in solution. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. This work successfully characterized the biophysical properties of AT in the presence of 2 emulsion adjuvants including a component study to rationalize how emulsion components affect protein antigen stability. [ABSTRACT FROM AUTHOR]

Cross-reacting material 197 (CRM 197 ), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM 197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM 197 molecules demonstrate that recombinant CRM 197 's expressed in heterologous systems ( Escherichia coli and Pseudomonas fluorescens ) are overall highly similar (if not better in some cases) to those expressed in the traditional system ( Corynebacterium diphtheriae ) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM 197 expressed in alternative sources have the potential to replace CRM 197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM 197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM 197 carrier proteins for use in various conjugate vaccines. [ABSTRACT FROM AUTHOR]

The worldwide switch to inactivated polio vaccines (IPVs) 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 Nanopatch™ (a solid, polymer microprojection 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 on 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 2 candidate tIPV formulations containing 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. [ABSTRACT FROM AUTHOR]

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C H 2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used. [ABSTRACT FROM AUTHOR]