Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.

The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use

Comparisons of optically monitored small-scale stirred tank vessels to optically controlled disposable bag bioreactors

<p>Abstract</p> <p>Background</p> <p>Upstream bioprocesses are extremely complex since living organisms are used to generate active pharmaceutical ingredients (APIs). Cells in culture behave uniquely in response to their environment, thus culture conditions must be precisely defined and controlled in order for productivity and product quality to be reproducible. Thus, development culturing platforms are needed where many experiments can be carried out at once and pertinent scale-up information can be obtained.</p> <p>Results</p> <p>Here we have tested a High Throughput Bioreactor (HTBR) as a scale-down model for a lab-scale wave-type bioreactor (CultiBag). Mass transfer was characterized in both systems and scaling based on volumetric oxygen mass transfer coefficient (k_La) was sufficient to give similar DO trends. HTBR and CultiBag cell growth and mAb production were highly comparable in the first experiment where DO and pH were allowed to vary freely. In the second experiment, growth and mAb production rates were lower in the HTBR as compared to the CultiBag, where pH was controlled. The differences in magnitude were not considered significant for biological systems.</p> <p>Conclusion</p> <p>Similar oxygen delivery rates were achieved in both systems, leading to comparable culture performance (growth and mAb production) across scales and mode of mixing. HTBR model was most fitting when neither system was pH-controlled, providing an information-rich alternative to typically non-monitored mL-scale platforms.</p

Comparison of exon 5 sequences from 35 class I genes of the BALB/c mouse

DNA sequences of the fifth exon, which encodes the transmembrane domain, were determined for the BALB/c mouse class I MHC genes and used to study the relationships between them. Based on nucleotide sequence similarity, the exon 5 sequences can be divided into seven groups. Although most members within each group are at least 80% similar to each other, comparison between groups reveals that the groups share little similarity. However, in spite of the extensive variation of the fifth exon sequences, analysis of their predicted amino acid translations reveals that only four class I gene fifth exons have frameshifts or stop codons that terminate their translation and prevent them from encoding a domain that is both hydrophobic and long enough to span a lipid bilayer. Exactly 27 of the remaining fifth exons could encode a domain that is similar to those of the transplantation antigens in that it consists of a proline-rich connecting peptide, a transmembrane segment, and a cytoplasmic portion with membrane-anchoring basic residues. The conservation of this motif in the majority of the fifth exon translations in spite of extensive variation suggests that selective pressure exists for these exons to maintain their ability to encode a functional transmembrane domain, raising the possibility that many of the nonclassical class I genes encode functionally important products

Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.

The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use

Prediction of N-linked Glycoform Profiles of Monoclonal Antibody with Extracellular Metabolites and Two-Step Intracellular Models

Monoclonal antibodies (mAbs) are commonly glycosylated and show varying levels of galactose attachment. A set of experiments in our work showed that the galactosylation level of mAbs was altered by the culture conditions of hybridoma cells. The uridine diphosphate galactose (UDP-Gal) is one of the substrates of galactosylation. Based on that, we proposed a two-step model to predict N-linked glycoform profiles by solely using extracellular metabolites from cell culture. At the first step, the flux level of UDP-Gal in each culture was estimated based on a computational flux balance analysis (FBA); its level was found to be linear with the galactosylation degree on mAbs. At the second step, the glycoform profiles especially for G0F (agalactosylated), G1F (monogalactosylated) and G2F (digalactosylated) were predicted by a kinetic model. The model outputs well matched with the experimental data. Our study demonstrated that the integrated mathematical approach combining FBA and kinetic model is a promising strategy to predict glycoform profiles for mAbs during cell culture processes

Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules

Process analytical technology for recombinant pandemic flu vaccines: Viral ultrastructure, aggregation, and binding

Here we describe our progress in developing electrospray differential mobility analysis (ES-DMA) into a process analytical technology (PAT) for recombinant vaccines. The threat of pandemic flu (e.g. avian influenza H5N1, swine flu H1N1, etc.) remains a significant public concern. Recombinantly produced vaccines hold significant advantages over traditional means of growing vaccines including increased production rates, shorter times between viral identification and clinical treatment, and improved safety profiles. However, accelerated production methods also require advanced process control sensors (i.e. process analytical technologies) that can rapidly and accurately detect out-of-specification intermediates. ES-DMA has the potential to fulfill this need. Here we describe our efforts to use ES-DMA to (1) rapidly quantify the assembly state and integrity of recombinantly produced virus like particles (VLPs), (2) determine the viral aggregation state, and (3) quantify antibody-virus binding necessary to evaluate stoichiometry and vaccine efficacy