Biology subjects

Stiving, A. Q.

Publications and source records attributed to Stiving, A. Q..

4 recordsLinked to original sources

bioRxiv2024

Systematic Optimization of Activity-Based Protein Profiling for Identification of Polysorbate-Degradative Enzymes in Biotherapeutic Drug Substance Down to 10 ppb

The identification and control of high-risk host cell proteins (HCPs) in biotherapeutics development is crucial for ensuring product quality and shelf life. Specifically, HCPs with hydrolase activity can cause degradation of excipient polysorbates (PS), leading to a decrease in the shelf life of the drug product. In this study, we systematically optimized every step of an activity-based protein profiling (ABPP) workflow to identify trace amounts of active polysorbate-degradative enzymes (PSDEs) in biotherapeutics process intermediates. Evaluation of various parameters during sample preparation pinpointed optimal pH level and fluorophosphonate (FP)-biotin concentration. Moreover, the combined use of a short liquid chromatography gradient and the fast-scanning Parallel Accumulation Serial Fragmentation (PASEF) methodology increased sample throughput without compromising identification coverage. Tuning Trapped Ion Mobility Spectrometry (TIMS) parameters further enhanced sensitivity. In addition, we evaluated various data acquisition modes including PASEF combined with data-dependent acquisition (DDA PASEF), data-independent acquisition (diaPASEF), or parallel reaction monitoring (prm-PASEF). By employing the newly optimized ABPP workflow, we successfully identified PSDEs at a concentration as low as 10 parts per billion (ppb) in a drug substance sample. Finally, the new workflow enabled us to detect a PSDE that could not be detected with the original workflow during a PS degradation root-cause investigation.

bioengineering↗

bioRxiv2024

Enabling functionality and translation fidelity characterization of mRNA-based vaccines with a platform-based, antibody-free mass spectrometry detection approach

The success of mRNA-based therapeutics and vaccines can be attributed to their rapid development, adaptability to new disease variants, and scalable production. Modified ribonucleotides are often used in mRNA-based vaccines or therapeutics to enhance stability and reduce immunogenicity. However, substituting uridine with N1-methylpseudouridine has recently been shown to result in +1 ribosomal frameshifting that induces cellular immunity to the translated off-target protein. To accelerate vaccine development, it is critical to have analytical methods that can be rapidly brought online to assess the functionality and translation fidelity of mRNA constructs. Here, a platform-based, antibody-free method was developed using cell-free translation (CFT) and liquid chromatography-tandem mass spectrometry (MS) that can detect, characterize, and provide relative quantification of antigen proteins translated from mRNA vaccine drug substance. This workflow enabled the evaluation of mRNA subjected to thermal stress as well as bivalent (i.e., two mRNA encoding different antigen variants) drug substance. Additionally, the MS detection approach exhibited high sensitivity and specificity by accurately identifying all six translated proteins and their relative abundances in a dose-dependent manner following transfection of human cells with a hexavalent mRNA mixture encapsulated in lipid nanoparticles (LNPs), despite significant protein sequence homology. Expanding on these efforts, we show the utility of the CFT-MS approach in identifying the presence and junction of +1 ribosomal frameshifting resulting from N1-methylpseudouridation. Overall, this CFT-MS methodology offers a valuable analytical tool for the development and production of mRNA-based vaccines by facilitating the evaluation of mRNA quality and functionality while ensuring accurate translation of antigen proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/594137v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4c7a7org.highwire.dtl.DTLVardef@1efddd1org.highwire.dtl.DTLVardef@cbfcbaorg.highwire.dtl.DTLVardef@1ae1acc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

diaPASEF Enables High-throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutics production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein, as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF), to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.

bioengineering↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗