CDER\u27s emerging technology team

The newly formed Emerging Technology Team (ETT) draws membership from all CDER quality review, research and inspection functions, including Office of Biotechnology Products. The goal of the ETT is provide a primary point of contact for external inquiries regarding emerging technology in pharmaceutical and biotechnology manufacturing and quality control. The ETT will partner with review offices in a cross-functional manner and identify regulatory strategy and resolve roadblocks to new technologies relating to existing guidance, policy or practices related to review or inspection. The ETT’s initial focus will be innovation on novel products, manufacturing processes, or testing technologies or processes to be submitted in a BLA, NDA or ANDA. This talk will provide an update on ETT’s activities in 2015

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

FDA/OBP laboratory research to support continuous bioprocessing

Recently, there has been a movement in commercial biotechnology drug production to develop fully a continuous manufacturing scheme capable of consistent production of high quality therapeutics. The FDA is increasingly reviewing applications implementing elements of continuous manufacturing or enabling technologies. This includes product and process engineering, and integration of enabling technology during development. Achieving a true continuous process can be difficult and raise additional unknown regulatory concerns (i.e. how to handle process interruptions or unmatched liquid flow rates between linked unit operations, how to measure viral clearance and establish safety). This poster will provide an overview of CDER’s, Office of Biotechnology Product’s lab capabilities and selected regulatory research case studies on continuous biomanufacturing and enabling technologies. These lab-based capabilities are being leveraged to study continuous bioreactor cell culture production, continuous chromatography, viral safety, and Process Analytical Technology (PAT) tools to enable these operations. Preliminary results have provided encouraging data to broaden technological challenges and potential benefits of continuous biomanufacturing approaches

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

Design of a novel continuous flow reactor for low pH viral inactivation

Currently the Biopharamaceutical industry is moving from operating in batch mode to continuous manufacturing. Low pH viral inactivation is a highly effective and a common method used in monoclonal antibody purification processes. During this unit operation the product is pooled and held, presenting a major bottle neck to end-to-end continuous downstream processing. Moving from a holding tank to a tubular reactor would provide for a means of processing materials continuously. The major challenges with tubular reactors for this application include limiting and characterizing the axial dispersion to ensure sufficient incubation time. The main objective of this work was to design and characterization of the residence time distribution (RTD), exit age of fluid elements leaving the reactor, of a continuous tubular reactor (CTR) for low pH viral inactivation. The following CTR design criteria were generated to streamline integration into the downstream purification process: (1) a ≤ 5 psi pressure drop along the length of the tube, (2) radial mixing within the reactor without moving parts to minimize axial dispersion, (3) a minimum residence time (MRT) approach was used to ensure that the desired product holding time was met, (4) operating at the laminar flow regime to limit shear on the product and minimize the pressure drop along the tube length while operating at flow rates sufficient for a 100 L bioreactor continuous process. Curved pipes offer improved radial mixing due to the formation of Dean Vortices via centrifugal forces. Thus, to reduce axial dispersion, the reactor as designed to include curvature in flow path via alternating 270 turns which also induced changes in the flow direction with each turn or flow inversions. A modular design with incubation chambers that can be connected in series was generated and evaluated using computation fluid dynamic (CFD) simulation before a final design was 3D printed and experimentally evaluated. Comprehensive computational fluid dynamics modeling in ANSYS Fluent of the CTR via velocity profile and secondary flow streamlines show enhanced radial mixing due to secondary flows and changes in flow direction. CFD simulation results were validated by pulsed tracer experiments and were in sufficient agreement, RTD variance values within 6.7%, with the computational model. Scaling the CTR with length to ~115.1 m at 50 ml/min, resulted in a MRT of 70.4 ± 0.46 mins with a pressure drop of ~0.7 psi. With increased length the dimensionless RTD profiles become more symmetrical and tighter about the mean residence time, indicating a smaller deviation from plug flow with increased length. Further scalability of the design is currently under investigation via generation and CFD analysis of a geometrical scale-down model for viral clearance studies

Analysis of Expression, Structure and Evolution of Non-Classical Class I Major Histocompatibility Complex Genes

Class I major histocompatibility molecules (MHC) are 45 kilodalton (kD) glycoproteins that associate with a smaller 12 kD polypeptide, β₂-microglobulin. In the BALB/c mouse, there are three classical class I molecules, H-2Kᵈ, Dᵈ, and Lᵈ, which are expressed throughout the body and present viral antigens to cytotoxic T lymphocytes (CTLs). In addition to the genes that encode the three classical class I antigens, the BALB/c genome contains 32 genes that structurally resemble the classical class I genes, and therefore possibly encode class I molecules. A few of the non-classical class I genes have been shown to encode molecules, TL, Qa-1, Qa-2, Q10, Qb-1, and Hmt, which are expressed in a generally tissue-specific manner, and probably do not act as restriction elements. However, it is unclear what function these molecules play, or why such a large gene family is maintained if only three viral antigen-presenting restriction elements are required by the murine immune system. DNA sequences were obtained from each of the 35 class I genes of the BALB/c mouse of the transmembrane domain-encoding fifth exon. Based on nucleotide sequence similarity, the fifth exons could be divided into seven groups that share little similarity with each other. In addition, the majority of the fifth exons are able to encode a transmembrane domain that can be separated into a proline-rich connecting peptide, a hydrophobic transmembrane segment, and a ctyoplasmic portion that includes basic anchoring residues. Since this conservation occurs in spite of extensive variation of nucleotide sequence in these exons, it is likely that selective pressure exists to maintain a functional structure in the majority of class I genes. A cDNA library was constructed from a thymus from a five-week-old BALB/c mouse. From this library, 69 class I cDNA transcripts from 15 different class I genes were isolated and analyzed. Included were three novel transcripts from Tla subregion genes, the T9ᶜ, T17ᶜ, and T18ᶜ genes. Sequence analysis of these clones reveals that the T9ᶜ gene is probably a pseudogene, while the T18ᶜ gene has an open reading frame in at least exons 2, 3, 4, and 5. A fourth cDNA clone was a transcript from the Thy19.4 gene, a gene that had not been previously isolated on a recombinant DNA clone. The isolation of transcripts from such a relatively large number of genes suggests that the number of expressed and perhaps functionally important class I genes may be larger than previously believed, and that expression of class I recognition structures may be important for cell-cell interactions within the thymus. To further pursue the characterization of the Thy19.4 gene, a genomic clone containing this gene was isolated from a size-selected insert library, and the DNA sequence of the Thy19.4 gene was obtained. The Thy19.4 gene contains an open reading frame, and in several aspects resembles the genes that encode the transplantation antigens. These similarities include a shared exon/intron structure and shared amino acid sequence motifs. In addition, PCR amplification experiments using tissue cDNA demonstrates that the Thy19.4 gene is expressed in a variety of tissues. However, unlike the classical transplantation antigens, the Thy19.4 gene maps distal to the H-2 region, in the Hmt region. These studies have demonstrated that class I gene transcription is more extensive than previously believed. Some of the expressed genes, like the T18ᶜ and Thy19.4 genes, appear to be able to encode class I molecules which may share structural characteristics with the classical transplantation antigens and may possibly serve as recognition structures in cell-cell interaction events. In addition, examination of the transmembrane domain exon of each of the 35 class I genes suggests that some selective constraint is acting on the majority of members of this family of genes, thus raising the possibility that many of the nonclassical class I genes encode functionally important products.</p

Hybridoma cell-culture and glycan profile dataset at various bioreactor conditions

This is an “11 factor-2 level-12 run” Plackett-Burman experimental design dataset. The dataset includes 11 engineering bioreactor parameters as input variables. These 11 factors were varied at 2 levels and 23 response variables that are glycan profile attributes, were measured “A Design Space Exploration for Control of Critical Quality Attributes of mAb” (H. Bhatia, E.K. Read, C.D. Agarabi, K.A. Brorson, S.C. Lute, S. Yoon S, 2016) [2]

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