Quantitation of strain-specific hemagglutinin trimers in mosaic quadrivalent influenza nanoparticle vaccine by ELISA.

An enzyme linked immunosorbent assay (ELISA) method was developed to analyze the assembly of a tetravalent mosaic influenza nanoparticle (NP) vaccine, Flumos-v1, consisting of hemagglutinin trimers (HAT) from H1 (A/Idaho/07/2018), H3 (A/Perth/1008/2019), HBV (Vic-B/Colorado/06/2017) and HBY (Yam-B/Phuket/3073/2013) strains. The sandwich ELISA assay used lectin from Galanthus nivalis as a universal capture reagent for all HAT strains and specific monoclonal antibody (mAb) to detect corresponding hemagglutinin antigen. The mAb binding of HATs incorporated into NPs diverged from those for single HAT solutions, resulting in inaccurate quantitation of assembled HATs. An optimized zwittergent treatment was used to fully dissociate the influenza NP and aligned binding activities in each pair of single HAT and dissociated HAT from NP. The dissociated HATs were then quantified against their corresponding HAT standard solutions for three development lots of FluMos-v1 vaccine and the assembly ratio of all four HATs was calculated. The molar ratio of different HATs incorporated into this quadrivalent NP vaccine was consistent and determined as H3:H1: HBV: HBY âˆ¼ 1.00:0.92:0.96:0.87, which was close the expected 1:1:1:1 ratio and confirmed a proper assembling of multivalent NP.

Site-Specific Fluorescent Labeling of Hemagglutinin-Specific Antigen Binding Fragment through Amine Chemistry Revealed by Mass Spectrometry.

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles.

Characterization of Flu MOSAIC nanoparticle vaccine candidate using high performance size-exclusion chromatography to support vaccine process development.

This report describes an application of analytical high performance size exclusion chromatography with UV and Fluorescent detection (HPSEC-UV/FLR) method that enabled a bridging from research vaccine candidate discovery (His-tagged model) to clinical product development (Non-His-tagged molecules). HPSEC measurement can accurately determine the total trimer-to-pentamer molar ratio by either titration evaluation during the nanoparticle being assembled or dissociation during a well-formed nanoparticle being dis-assembled. Through experimental design with small sample consumptions, HPSEC can provide a quick determination on the nanoparticle assembling efficiency which can therefore guide the buffer optimization for an assembly, from His-tagged model nanoparticle, to non-His-tagged clinical development product. HPSEC has also discovered a difference in assembling efficiencies for various strains of HAx-dn5B with Pentamer-dn5A components, and different efficiencies for monovalent assembly vs. multivalent assembly. The present study demonstrates HPSEC as a pivotal tool to support the Flu Mosaic nanoparticle vaccine development from research to clinical production.

Development and validation of a mass spectrometry based analytical method to quantify the ratios in hemagglutinin trimers in quadrivalent influenza nanoparticle vaccine - FluMos-v1.

A quadrivalent influenza nanoparticle vaccine (FluMos-v1) offers long-lasting protection against multiple influenza virus strains and is composed of four strains of hemagglutinin trimer (HAT) assembled around a pentamer core. Here we report an LC-MS/MS analytical development and validation method to measure the percentage of each HAT component in FluMos-v1.

Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development.

Broadly neutralizing antibody (bNAb) CAP256-VRC26.25 (abbreviated CAP256LS), a human IgGI monoclonal antibody targeting the V1V2 site of the HIV-1 envelope, has demonstrated high therapeutic potential as a broadly neutralizing monoclonal antibody against HIV-1. During the process development, a heavy chain fragmentation (clipping) was observed, that led to a relative potency reduction. In this report, we highlighted a series of process and product mitigation strategies deployed to advance this product. We have detailed how analytical characterization tools, especially the microchip reduced capillary gel electrophoresis (CGE-SDS), played a pivotal role in identifying the development issues and in providing measurements to guide implementation of mitigation strategies.

Tyrosine O-sulfation proteoforms affect HIV-1 monoclonal antibody potency.

CAP256V2LS, a broadly neutralizing monoclonal antibody (bNAb), is being pursued as a promising drug for HIV-1 prevention. The total level of tyrosine-O-sulfation, a post-translational modification, was known to play a key role for antibody biological activity. More importantly, here wedescribe for the first time the significance of the tyrosine-O-sulfation proteoforms. We developed a hydrophobic interaction chromatography (HIC) method to separate and quantify different sulfation proteoforms, which led to the direct functionality assessment of tyrosine-sulfated species. The fully sulfated (4-SO3) proteoform demonstrated the highest in vitro relative antigen binding potency and neutralization efficiency against a panel of HIV-1 viruses. Interestingly, highly variable levels of 4-SO3 were produced by different clonal CHO cell lines, which helped the bNAb process development towards production of a highly potent CAP256V2LS clinical product with high 4-SO3 proteoform. This study presents powerful insight for any biotherapeutic protein development where sulfation may play an important role in product efficacy.

Quantification of prefusion conformation for HIV vaccine using size-exclusion chromatography.

A closed prefusion conformation or an open (non-prefusion) conformational state of a protein vaccine candidate molecule can determine if it effectively elucidates a desired immunity. A quick and reliable method to monitor conformational state is important during vaccine development. In addition to our existing immunoassays, we have developed a unique physicochemical approach using size-exclusion chromatography to assess binding between antibody and the structurally desired antigen protein. Through the bound monoclonal antibody protein vaccine peak shift in the size-exclusion chromatography profile, this method determines the percent closed (prefusion) conformation present in a sample. Since only the closed prefusion conformation binds to the specific antibody, the population of the closed versus the open conformation of the vaccine molecule can be monitored without the need for a reference calibrator. This new method can be applied broadly to vaccine development, as well as for antibody selection during antibody drug discovery. The mAb CAP256V2LS (250 µg/mL) specific to prefusion conformation was mixed with HIV trimer (250 µg/mL) at 2:1 volume ratio, incubated at 37 °C for 30 mins and injected onto HPLC column. The percent of non-prefusion conformation was calculated based on ratio of peak area of unbound trimer and total area of control trimer sample (without mAb).

Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods.

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

Development and application of an analytical approach to assess an antibody's potential for disulfide reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction has been observed in a recent large-scale clinical manufacturing operation. A massive reduction/precipitation at post-clarification steps has occurred. This note presents the development of a novel analytical approach to identify the "potential reduction"-a unique approach to predict the propensity of a monomeric-profiled mAb to be reduced in the post-harvest stage, such as harvest clarification and/or purification steps. The core of this new approach includes comparing the non-reducing capillary electrophoresis profiles of pre- and post-vacuum treated mAb in harvest cell culture fluid (HCCF). Using this approach, the potential reductions of two in-house mAbs in the unclarified and clarified cell culture harvest were assessed.

Streamlining Peptide Mapping LC-MS Approach for Studying Fusion Peptide-Conjugated Vaccine Immunogens.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MSE fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

Advances in Purification of SARS-CoV-2 Spike Ectodomain Protein Using High-Throughput Screening and Non-Affinity Methods.

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics.

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.

Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Protein and glycan molecular weight determination of highly glycosylated HIV-1 envelope trimers by HPSEC-MALS.

High Performance Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering detection (HPSEC-MALS) is an important tool to provide a reliable molecular weight measurement for a large complex biomolecule. A recent HIV-1 soluble envelope trimer vaccine candidate, BG505 DS-SOSIP.664, is among the most glycosylated proteins to enter a clinical trial to date, and determination of its protein and glycan molecular weight is one of the key attributes in pre-clinical characterization. However, protein and glycans possess disparate dndcvalues making molecular weight measurement inaccurate in conventional SEC-MALS. To overcome these challenges, a simple mathematically guided experiment was explored, and a composite dndcvalue was established by utilizing protein and glycan mass contributions for the HIV-1 envelope trimer. This establishment was further verified by an orthogonal mass spectrometry analysis. This innovative, simple, and quick analytical approach can be applied broadly to measuring the molecular weight of various composite molecular structures, such as complex glycoconjugates.

Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelope glycoprotein.

One of the HIV-1 vaccine design efforts has focused on developing a recombinant HIV-1 trimeric envelope glycoprotein (Env) as an immunogen to induce broadly neutralizing antibodies. A native-like immunogen, the BG505.DS.SOSIP.664 gp140 (Env) construct has been well-characterized as a vaccine candidate. This vaccine candidate comprises of three identical gp120 and truncated gp41 subunits that form into a trimer of heterodimers. During production, recombinant Env is expressed as a gp140 precursor polypeptide in which a furin cleavable site is engineered to generate a heterodimer of gp120 and gp41 subunits. Each heterodimer is connected by an intermolecular disulfide bond, and three heterodimers form into a trimer. Furin cleavage is an important factor to mimic native-like HIV-1 Env conformations and is needed to help induce an immune response. Therefore, it is critical to monitor cleavage for ensuring functionality of the Env vaccine product. In this paper, a new RPLC-UV method coupled with reduction was developed to routinely determine the percentage of uncleaved gp140 relative to the cleaved gp120 and gp41 subunits. Baseline separation was achieved among the gp120, gp41 and uncleaved gp140 peaks, thus enabling relative quantification of uncleaved gp140. Overall, this RPLC-UV approach has been successfully applied to support Env vaccine candidate developments.

A non-affinity purification process for GMP production of prefusion-closed HIV-1 envelope trimers from clades A and C for clinical evaluation.

Metastable glycosylated immunogens present challenges for GMP manufacturing. The HIV-1 envelope (Env) glycoprotein trimer is covered by N-linked glycan comprising half its mass and requires both trimer assembly and subunit cleavage to fold into a prefusion-closed conformation. This conformation, the vaccine-desired antigenic state, is both metastable to structural rearrangement and labile to subunit dissociation. Prior reported GMP manufacturing for a soluble trimer stabilized in a near-native state by disulfide (SOS) and Ile-to-Pro (IP) mutations has employed affinity methods based on antibody 2G12, which recognizes only ~30% of circulating HIV strains. Here, we develop a scalable manufacturing process based on commercially available, non-affinity resins, and we apply the process to current GMP (cGMP) production of trimers from clades A and C, which have been found to boost cross-clade neutralizing responses in vaccine-test species. The clade A trimer, which we named "BG505 DS-SOSIP.664", contained an engineered disulfide (201C-433C; DS) within gp120, which further stabilized this trimer in a prefusion-closed conformation resistant to CD4-induced triggering. BG505 DS-SOSIP.664 was expressed in a CHO-DG44 stable cell line and purified with initial and final tangential flow filtration steps, three commercially available resin-based chromatography steps, and two orthogonal viral clearance steps. The non-affinity purification enabled efficient scale-up, with a 250 L-scale cGMP run yielding 9.6 g of purified BG505 DS-SOSIP.664. Antigenic analysis indicated retention of a prefusion-closed conformation, including recognition by apex-directed and fusion peptide-directed antibodies. The developed manufacturing process was suitable for 50 L-scale production of a second prefusion-stabilized Env trimer vaccine candidate, ConC-FP8v2 RnS-3mut-2G-SOSIP.664, yielding 7.8 g of this consensus clade C trimer. The successful process development and purification scale-up of HIV-1 Env trimers from different clades by using commercially available materials provide experimental demonstration for cGMP manufacturing of trimeric HIV-Env vaccine immunogens, in an antigenically desired conformation, without the use of costly affinity resins.

Development of a High Yielding Bioprocess for a Pre-fusion RSV Subunit Vaccine.

Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).

Quantitation of residual valproic acid in flu vaccine drug substance.

Quantitative measurement of process-related impurities is a critical safety requirement for the production of drug substances of vaccine and therapeutic biologics. A simple and sensitive HPLC method has been developed for separation and quantitation of residual valproic acid (VPA) used in the cell transfection procedure for the manufacturing of an influenza vaccine. The method is comprised of a modified Dole liquid phase extraction followed by a quick pre-column derivatization using 2-bromoacetophenone. Nonanoic acid (NNA) is used as the internal standard (IS) and the quantification is performed by reversed-phase liquid chromatography. This new method can accurately measure as low as 6.8 µg/mL (LOQ) residual VPA in the vaccine drug substance.

Rationally designed coiled-coil DNA looping peptides control DNA topology.

Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.