Publications by authors named "Daniel B Gowetski"

Article Synopsis
  • Bi-functional N-Hydroxysuccinimide (NHS) linkers are crucial in connecting immunogens with carrier proteins to enhance immune responses, particularly in HIV-1 vaccine development.
  • The study investigated the degradation kinetics of these linkers using reversed-phase liquid chromatography (RPLC-UV) to ensure effective conjugation before the linkers become inactive.
  • Three types of cross-linkers were examined for their degradation pathways, with specific kinetics reported for the Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB) linker.
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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.

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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 -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.

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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.

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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.

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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.

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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.

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Article Synopsis
  • CAP256V2LS is a monoclonal antibody being developed for HIV-1 prevention, highlighting the importance of tyrosine-O-sulfation in its biological activity.
  • The researchers introduced a new chromatography method to analyze and quantify different sulfated proteoforms, discovering that the fully sulfated form (4-SO) was the most effective in binding to and neutralizing HIV-1 viruses.
  • Variations in the production of the 4-SO proteoform from different CHO cell lines are important for optimizing the development of a potent clinical product, emphasizing the role of sulfation in enhancing the efficacy of biotherapeutics.
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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.

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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.

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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.

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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.

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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.

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Article Synopsis
  • The research focuses on using recombinant Tetanus toxoid heavy chain fragment C (rTTHC) as a carrier to enhance the effectiveness of HIV-1 fusion peptide vaccines.
  • Comprehensive mass spectrometry was conducted during rTTHC purification, revealing three types of cysteine modifications linked to the protein's oxidative states.
  • These findings helped confirm the identity of the rTTHC protein, ensuring the purification process was effective and laying the groundwork for further vaccine development.
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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.

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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.

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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.

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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.

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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.

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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.

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