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THE Industry Meeting Place to Exchange Real-World Solutions to Improve Speed, Cost and Quality

Conference: October 26-29, 2015 · Exhibition: October 17-19, 2015 · John B Hynes Veterans Memorial Convention Center · Boston, MA

2014 Poster Presentations

2014 Poster Presentations

Learn new approaches to your research programs
Below is a list of the scientific poster presentations accepted for display* at BioProcess International™ Conference and Exhibition 2014. (Submit a poster abstract.)

Updated: October 2, 2014

Analytical & Quality

A1 Quality Risk Assessment, Quality Auditing and Product Lifecycle Planning Karen Bossert, Lyophilization Technology, Inc. Clinical manufacturing occurs during the earliest stages of product development, when little is known about the API, its impurities and degradation products. Innovator firms have a distinct challenge in deciding where to set the bar for regulatory compliance at each step along the development pathway, with quality requirements typically becoming more stringent as the product progresses in the development life cycle. For Contract Manufacturing Organizations (CMOs), balance must be achieved between the flexibility required to meet the needs of each client and maintaining firm internal quality standards necessary to establish the state of control required for manufacture of clinical supplies. The principles of Quality Risk Management can be applied to design and monitor appropriate quality systems for clinical manufacturing. Application of these principles provide flexibility for a range of customer quality standards and products, while allowing the CMO and customer to meet the regulatory needs of a global supply environment. Using experience as a CMO focused on sterile products, data were compiled from customers ranging from small innovator firms to established, multi-national companies, each with unique perspective and approaches to meeting regulatory requirements for clinical supplies at each phase. Principles of risk analysis were used to identify areas where rigid infrastructure was required to maintain an appropriate state of control in manufacturing operations. Site quality system metrics were compiled and inherent challenges of early phase clinical manufacturing were identified. The resulting best practices were aligned with regulatory requirements of markets served and implemented throughout the organization.

A2 A comparison of Methods for Quantifying Silicone Droplets in Biologics Using Dynamic Imaging Particle Analysis Lew Brown, Fluid Imaging Technologies, Inc. Dynamic Imaging Particle Analysis (DIPA) is increasingly being used as a method of characterizing sub-visible particulates in protein-based therapeutics. Many of these formulations, particularly pre-filled syringes, will contain silicone droplets which are used for lubrication. When reporting particulate content, especially in the 2µm to 10µm size range, it is desirable to eliminate the contribution of the silicone droplets to the overall particle count, since they are considered necessary and non-harmful. Since DIPA can measure particle shape parameters, it can potentially be used to "filter out" the silicone droplets prior to reporting particle count. This presentation will look at several different mathematical approaches to identifying and quantifying the silicone droplets in the sample. A data set will be acquired with images from a sample protein therapeutic containing both protein aggregates and silicone droplets. The data set will first be analyzed visually to make a baseline determination of the number of protein aggregates and silicone droplets. This baseline data set will have an assigned "particle type" value given to every particle image collected. This same original data set will then be subjected to automated, algorithmic analysis using different methods to try to quantify the silicone droplet content. For each different algorithm or method applied, the total number of silicone droplets identified by the method will be recorded, and the number of "false positives" (if any) will also be recorded by comparison to the baseline manually classified data set.

A3 A High Throughput Formulation Platform for Monoclonal Antibodies: Development and Application Paul Casaz, Massbiologics of the University of Massachusetts Medical School A successful formulation for a monoclonal antibody (MAb) requires evaluation of a large number of potential candidates for their ability to minimize chemical changes, fragmentation and aggregation of the product during accelerated stability studies. We have developed a systematic high throughput platform to evaluate protein stability in a range of formulations in 96 well plates. The formulations are prepared in a deep well template by adding a protein solution to concentrated formulation buffers. Replicate plates made from this template can then be exposed to different stress conditions. The use of high precision assays (SE-UPLC, DSF, turbidity, solubility, extrinsic fluorescence, and capillary zone electrophoresis) allows physical and chemical changes to the antibody to be detected within one month at 37°C. Small amounts of sample are required for these assays so the screening can be done early in development when the amount of product available is limited. Examples of how this screening platform has been applied to MAb formulations will be presented. Comparison of the data generated by the screening method with real time stability is used to evaluate the utility of the screening assays.

A4 A case study in overcoming challenges in supporting a post-launch manufacturing site transfer of T-DM1/Kadcyla® drug product Lan Dai, Genentech/Roche Following the recent global approvals of T-DM1/Kadcyla® to treat metastatic HER2-positive breast cancer, a strategy has been developed to introduce post-launch manufacturing changes to ensure a robust supply-chain. T-DM1 is an antibody-drug conjugate from Genentech/Roche and is a member of a relatively new class of bio-therapeutic (with only one other currently approved product) where there is little regulatory precedent on post-approval changes. Herein we report our most recent experience in supporting a manufacturing site transfer of T-DM1/Kadcyla® lyophilized Drug Product from a contract manufacturing organization to an internal facility. The lack of precedents for post approval changes to ADC manufacturing resulted in challenges in designing process validation and comparability studies to demonstrate comparable product quality for a marketed therapeutic. For example, vaporized hydrogen peroxide (VHP) is used at the recipient site, but not the originating site, to sterilize fill lines. As a result, a VHP spiking study was required to assess the impact of different levels of residual VHP on Drug Product quality/stability and to define VHP limits for process validation. As part of the study, extended characterization, using LC-MS based peptide map, was performed to measure the individual oxidation level of critical methionine residues, which is the key to establish the acceptance limits for residual VHP level. For designing the comparability assessment, major inputs included (1) determining the critical quality attributes that might be impacted by the Drug Product manufacturing process, (2) deriving statistically-defined quantitative acceptance criteria based on manufacturing history, and (3) identifying appropriate assays for qualitative comparisons to address potential impacts of any differences between the process run at the two sites.

A5 A semi-automated, high-throughput methodology to characterize the N-glycosylation profile of a monoclonal antibody using the Agilent AssayMAP Bravo Elizabeth Dodson, BD Advanced Bioprocessing Protein quality is a primary concern for manufacturers of bio-therapeutics. Protein modifications with greatest impact on therapeutic function are known as critical quality attributes (CQA). The CQA's of a protein can be impacted at several stages of process development including clonal selection, cell culture, protein purification, and final formulation and storage. It is critical for us to have the capacity to assess the impact media and supplements have on protein quality. Here we describe the use of an automated, high-throughput methodology for the purification of monoclonal antibodies from cell culture supernatants with good yields. Further, the same technology was used in the semi-automated isolation and purification of N-glycans with comparable results to a "manual" methodology. The automated methodology allows for purification and characterization of up to 96 samples in under 4 hours as opposed to manual method (5 samples in three days). Taken together, the new high-throughput methodology provides a means of screening large numbers of cell culture conditions for the characterization of protein quality.

A6 Identifying Host-Cell Protease Responsible for Truncation of a Recombinant Protein Ruth Frenkel, Biogen Idec Variations in the cell culture media components can alter the expression of host-cell proteases, which in turn can impact biopharmaceutical product quality. A batch of recombinant biopharmaceutical Protein X failed release specification for charged isoforms distribution. Specifically, an increase of a lower pI truncated variant of Protein X was observed compared to historical batches from the manufacturing process. A subsequent root cause investigation narrowed the focus to cell culture operations, specifically associated with certain media powder lots, which may have enhanced expression or activity of certain host-cell proteases. A database search (CutDB.org) was performed to find proteases capable of cleaving at the amino acid residues consistent with the C-terminal truncation site of Protein X. A family of enzymes (Proteases XYZ family) was found that contained several capable of cleaving between the specific amino acid sequence motif of the observed truncation site. Various members of Proteases XYZ family were spiked into Protein X and fresh cell culture media. Spiked samples were analyzed by Strong Anion Exchange (SAX) chromatography to determine if any of the Proteases XYZ were able to cause an increase in the relative amount of the Protein X C-terminal truncated isoform. The results showed that a single member of the Protease XYZ family (Protease X) was a root cause for the increased truncated variants of Protein X.

A7 PetriJet - Innovative lab-automation solution for culture dish handling, imaging and automatic image analysis Felix Lenk, TU Dresden Petri dishes are a user-friendly and easy method for in vitro cultivation of not only plant cell and tissue cultures. Specific growth parameters, productivity and the influence of agar are only a small set of parameters which can be investigated by using petri dishes for cultivation. In larger screening experiments the manual and mostly only qualitative analysis is limited by the number of laboratory staff. Based on the constant development in laboratory automation an innovative solution to these problems is now for the first time readily available even for small and middle scaled laboratories with special requests due to their individual research profiles. With the presented fully automated, bench-top solution towards the handling of culture dishes, the experimental efforts can be reduced significantly and the sample throughput can be increased. For a continuously monitored, systematic, non-destructive and quantitative analysis of the culture dishes an automatic image recognition and analysis has been implemented. Any specific requirement for the analysis can be implemented into the algorithm. For long term experiments every culture dish gets labeled with a QR-code. With this system the culture dish can be tracked immediately by the algorithm and the new results can be displayed with respect to previous images. The precise analysis delivers reproducible results. These might also be used for quality assurance and analytical purposes. The laboratory bench-top device PetriJet can analyze up to 100 culture dishes within one hour. The user interaction for PetriJet handling is limited to starting, loading and unloading the device with a set of 20 petri dishes each. Currently, with dimensions of 800 x 600 x 400 mm the PetriJet is designed especially for small and medium scaled laboratories and reveals an ideal possibility for systematic and quantitatively analysis of samples on culture dishes.

A8 Optimization of CHO cell culture medium to target a monoclonal antibody charge variant profile Justin McGrath, BD Advanced Bioprocessing Protein quality of a biological therapeutic can be impacted at several stages of process development including clonal selection, cell culture, protein purification, and final formulation and storage. For BDAB understanding how cell culture media impacts protein quality is of paramount importance. Here we describe a two tiered approach for targeting the charge variant profile of a monoclonal antibody (mAb) expressed in a CHO K1 cell line. An existing animal component free (AF) basal medium did not produce a charge variant profile comparable to the desired profile of a reference protein. As a first strategy the basal media was reformulated to either remove or add certain components. The second approach was a new media screen and DOE around the optimal media with charge variant as one of the guiding parameters. Both strategies resulted in a much improved charge variant profile for the monoclonal antibody.

A9 Tracing of Extractables/Leachables through a mAb process using Mobius® ClinicReady Solutions Platform Hyesun McNulty, EMD Millipore The Mobius® ClinicReady Solutions is an innovative single-use platform that provides flexibility to build an entire upstream and downstream process, ranging from buffer /media preparation, clarification, virus filtration and TFF. One of major components of such single use systems is containers (bioreactor and buffer storage bags) which is constructed with polymer based material, hence has potential release of additional extractables/leachables into the final products compared to their traditional counterparts such as glass and stainless steel. These components are gamma irradiated as a mean of sterilization. There is a large body of studies evaluating extractables from various materials, but these studies have insufficient information on how extractalbes are added or removed throughout the process. We conducted a model study to trace extracables/leachables using a typical mAb conditions. A cell culture media (without inoculation) and buffer solutions were processed, collected and analyzed for extractables at nine major process points through Mobius Clinicready solution platform. The results of such study will be presented in this paper.

A10 Initial characterization of a novel cysteine protease for antibody sub unit analysis Fredrik Olsson, Genovis AB The IdeS enzyme (FabRICATOR) has emerged as a powerful tool for rapid characterization of antibody structural verification. Due to the extremely high specificity and favorable reaction conditions the combination of IdeS with with high-resolution mass spectrometry allows for rapid characterization of many critical quality attributes. Recently we identified another cysteine protease, which also proved to have a very high specificity. This cysteine protease digest human IgG1 above hinge generating two Fab fragments and one Fc fragment. Combining this new enzyme with IdeS enzyme allows for specific isolation of the complete hinge region of IgG1 in a non-reduced state with very high yield and accuracy.

A11 Single-use disposable sterile containers for Bioprocess Operations Ven Raman, EMD Millipore Biopharmaceutical companies are using single-use disposable sterile containers and assemblies for upstream and downstream processes at an ever increasing rate as these specialized technologies and products enable efficient and scalable processes. The newly developed PureFlex Plus™ film contains many of the same ultra low density polyethylene, poly(vinyl acetate) and poly(vinyl alcohol co-ethylene) layers that are in current PureFlex™ film. The only modification is the outer layer, to provide enhanced mechanical strength, creep resistance and limited permeability to air and gases. Since the process fluids contact the inner film layer, there is a need for understanding extractables from such containers at room and elevated temperatures and over prolonged time of exposure. For this purpose EMD Millipore conducted an extractable study on newly developed PureFlex Plus™ film and current PureFlex™ film A model stream approach was performed instead of conducting in actual formulations to minimize the analytical issues. The model streams selected for this study were water, 50% ethanol, 1N hydrochloric acid, 1N sodium hydroxide and 10% dimethyl sulfoxide. The results of this study will be presented. Authors: Ven Raman Ph.D, Paul Killian Ph.D, Touria Soukrathi M.S, Hyesun McNulty Ph.D EMD Millipore, Bedford MA

A12 Implementation of variable path length technology at Boehringer ingelheim Ramsey Shanbaky, C Technologies, Inc Absorption spectroscopy is used to quantify biomolecules using Beer's Law. At high concentrations, samples must be diluted due to limitation of traditional spectroscopy instrumentation utilizing a fixed path length of 1cm. A gravimetric correction is applied to ensure the accuracy of dilution. The process of diluting, applying the gravimetric correction and washing the cuvette is time consuming and can take several hours in a controlled setting such as Quality Control or Manufacturing. The use of variable path length technology eliminates the need to dilute samples by taking the absorbance measurements at multiple path lengths using a disposable optical fibrettte. The software plots absorbance vs. path length. Using a linear regression, the software calculates a slope for the points and determines the concentration by applying Beers Law. Use of variable path length technology for concentration measurements results in time savings of greater than 80%.

A13 Aggregation of Antibody-Drug Conjugates: The Light Scattering Toolbox for Screening and Characterization Daniel Some, Wyatt Technology Corp. Antibody-Drug Conjugates (ADCs) show much promise as effective therapeutics for cancer and other diseases. However, they often exhibit an increased aggregation propensity compared to their unmodified counterparts due to non-specific interactions arising from attached drug and linker moieties. Light scattering offers multiple techniques for addressing the challenges of formulation screening, and characterizing both aggregates and propensity for aggregation. We present these tools in the context of ADCs and the dependence of aggregation profiles on linker chemistry.

A14 Fast and Easy Generic Anti-CHO HCP Analysis, 96-Samples Assay-To-Data in 65 min Craig Tin, Pall Life Sciences Pall ForteBio has teamed up with Cygnus Technologies to jointly develop an Anti-CHO HCP detection kit. While ForteBio Octet systems are the industry standard in easy and rapid high throughput analysis, Cygnus HCP ELISA kits are known for their broad HCP recognition and sensitivity. The new ForteBio-Cygnus Anti-CHO HCP kit will embody the best of both worlds. Users will achieve unparalleled time-to-results, streamlined and automated workflow, enhanced dynamic range, and excellent precision and assay robustness.

A15 Determination of the Domain-Specific Degradation Profiles of SpeB Digested Rituximab Biosimilars by Imaged Capillary Isoelectric Focusing (iCIEF) Zichuan Zhang, PPD In this presentation, SpeB digestion coupling with iCIEF was developed for the determination of domain-specific degradation profiles of innovator and generic Rituximab (Rituxan, Mabthera and Reditux). Untreated and base treated (pH 10 at 40 ?C for 24 h) Rituximab samples were digested by SpeB ("FabULOUS", Genovis, Sweden) in reduced condition for 1 h at 37 ?C to obtain Fd, Fc/2 and light chain (LC), and mixed with ampholyte solution consisting of 2% of Pharmalyte 5-8, 2% of Pharmalyte 8-10.5, 0.5% of pI marker 5.85, 0.5% of pI marker 10.45, 0.35 % of methylcellulose and 7 M urea. An iCE3 instrument with UV detection at 280 nm (Protein Simple, CA) was used and Empower 2.0 software (Waters, MA) was used for data analysis. It was observed that untreated Rituxan (GenenTech, US) and Mabthera (Roche, EU) samples shared identical peak distribution and charge heterogeneity, while multiple charge variations of Fc/2 and less acidic Fd species were found from Reditux (Dr. Reddy, India). After 24 h treatment in pH 10, similar degradation products were observed from all three Rituximab samples. These results indicate that the established method can be applied to the domain-specific characterization of biosimilar therapeutic products.

A16 High Throughput Methods Evaluation for Impurities Determination during In-Process Development Li Zhang, Boehringer Ingelheim Host Cell Protein (HCP) and leached Protein A (ProA) are bioprocess-related impurities that are derived from the cell culture and antibody production using Protein A or MabSelect affinity chromatography. Monitoring the removal of these impurities in intermediate and final products is a regulatory requirement. Traditionally, ELISA methods have been used to determine the amount of HCP and ProA impurity levels in the antibody products. Those are often labor-intensive, with a narrow dynamic range and limited sample throughput. Gyrolab, Solid Phase Proximity ligation assay (SP-PLA) and Octet are different new technologies that can be used to determine the HCP and ProA levels. Here we discuss the comparison of these methods to ELISA for the quantification of host cell proteins and leached Protein A during antibody production.

Cell Culture and Upstream Processing

B1 A Novel System to enhance the Production of Erythroid Cells from Human Induced Pluripotent Stem Cells using HIF-1α NIHAL ALMURAIKHI, IMPERIAL COLLEGE LONDON Blood shortage is one of the major global concerns as there is a significant imbalance in donations comparing to transfusion demands, which encouraged researchers to develop appropriate substitutes for donated blood using pluripotent stem cells. There has been a rising excitement lately that human induced pluripotent stem cells (iPS cells) could provide patient-specific cells for cellular therapy in addition to their differentiation capability into any cell type, which can be exploited in erythroid cell production. Erythropoiesis is the process of making erythrocytes that can be enhanced by hypoxia-inducible factors 1-alpha (HIF-1α), a transcription factor known to facilitates cellular adaptation to hypoxia by over-expressing specific genes and stimulating many metabolic processes, including erythropoiesis, and angiogenesis. In this study, we have established a novel protocol to generate erythroid cells from human iPS cells using HIF-1α as a key enhancer. Beside the use of the standard cytokine cocktail used for erythroid induction; Epo, SCF, FLT3, TPO, IL3, and IL6, other growth factors were used; BMP4, VEGF, FGF, in addition to 5% serum. Supplementing the cells in 2D culture system with our novel optimized concentrations of the said cocktail under hypoxic condition showed the highest yield erythroid markers, mainly CD235a. Further maturation of those cells is required in order to achieve fully mature and functional RBCs phenotype. These results must be supported by the detection of Rh type and ABO grouping to ensure the presence of RBCs antigens. Eventually, after optimizing an enucleation protocol, globin detection and O2 dissociation curve must be also made to ensure functionality of the hemoglobin (incomplete work). Thus, considering all the above, the ultimate aim of this study is the efficient production of mature and functional RBCs in vitro from patient-specific iPS cells using HIF-1α to facilitate erythroid progenitor maturation and proliferation.

B2 Cell Culture Process Development for Fed-Batch Using a Novel Nutrient Additive Shawn Barrett, Thermo Fisher Scientific Bayer CHO cell-based process development for mAb production across 3 different host cell lines and 7 different projects has been summarized. A novel nutrient additive was extensively evaluated in 6 out of the 7 projects. By implementing the new platform process, high productivity was easily achieved in addition to desirable process attributes. The simple platform process also enabled the rapid transfer of antibody projects into clinical manufacturing and was demonstrated to be a robust, reproducible, and GMP-friendly fed-batch process.

B3 Continuous CHO Cell Culture Perfusion Platform for Process Development and Manufacturing Olivier Berteau, Charter Medical, Ltd. Bioreactor design is a complex engineering task requiring fine-tuning of bioreactor design and operational configuration to enable the cultured organisms to achieve their desired function. Before large-scale commercial manufacturing can be implemented, it is typically necessary to scale up a bioreactor during a process development stage. However, conventional scale-up techniques are fraught with technical challenges and financial risks. Perfusion bioreactor system in which a plurality of modular perfused bioreactors is operated in parallel to produce and maintain a biological cell culture is presented. In order to prevent contamination and facilitate segregation of particular peripheral bioreactors, the distributed system is configured such that one-way fluid communication is established from the central bioreactor to each of the peripheral bioreactors while maintaining fluid isolation among the peripheral bioreactors. In accordance with one exemplary non-limiting embodiment, a distributed perfusion bioreactor system is provided for producing and maintaining a continuous biological cell culture. The modularity of the peripheral bioreactors enables a scale-up free design of an exemplary bioreactor system that avoids the disadvantages of a conventional scale-up technique. The modularity of the peripheral bioreactors enables detection of process deviations and contamination, and easy and efficient segregation or restart of an affected peripheral bioreactor branch without disrupting the entire system. A distributed bioreactor arrangement is presented to maintain the cell culture continuously for extended periods of time using a commercially available CHO cell line.

B4 An Automated Bioreactor Sampling Solution for Assuring On-line PAT Analytical Fidelity Michael Biksacky, Flownamics, Inc. In the current bioprocessing environment a significant unmet need still exists for automated, precise, real-time analysis of important culture process parameters such as nutrients, metabolites and various cell measurements. Transforming traditional off-line analytical methods into effective, automated, on-line process analytical technology (PAT) platforms requires rapid and precise analysis that performs as good as or better than the manual off-line analytical method. An automated, on-line bioreactor sampling system was evaluated for its ability to preserve the analytical integrity of various integrated 3rd party off-line analyzers typically used for culture process monitoring. The performance evaluation encompassed a broad range of analytes and measurement ranges, while comparing automated on-line analysis with the analyzer’s respective manual, off-line analytical method. In short, the analytical fidelity of each integrated analyzer was preserved by the automated on-line sampling system, which demonstrated a reliable, automated on-line PAT solution for real-time bioreactor culture monitoring and control.

B5 Using Cell Response Dynamics Generated from Perfusion Reactor Studies to Optimize Fed Batch Bioreactor Productivity Jeff Breit, Bend Research Understanding and quantifying cellular dynamic responses to input process variables in bioreactors can facilitate the development of rational control schemes for fed batch and perfusion bioreactors. Often what is required to understand and quantify these dynamic responses are time course data sets that quantify the kinetic responses of cells to changes in process conditions, and an experimental methodology that excites the process dynamics in a manner which is relevant to a manufacturing environment. We have completed a cellular dynamic response study using a perfusion bioreactor system with the goal of identifying the process input ranges that positively impact cellular and protein quality attributes. This study modulated a key input variable important for glycosylation control and impacted sugar residues in a controlled fashion on a model monoclonal antibody. We identified an optimal working concentration range of the variable in our system for our model CHO cell line and used time-course cell specific rates, metabolic fluxes, and protein quality measurements to train a predictive model for protein quality. These modeling techniques were then used to identify methods to translate our findings to a fed batch system, required for adoption in many biomanufacturing settings.

B6 A Universal CD Feed for High Performance in a Bio-Production Process Kaci Conaway, Becton and Dickinson Optimization of cell culture parameters is a vital and challenging part of biotherapeutic process development. One such challenge is the selection of a scalable and appropriate cell culture feed that can work in combination with a given base medium to achieve the desired titer and growth characteristics. The typical approach is to screen multiple commercially available feeds and identify the most appropriate feed for the production process with a specific base medium. However, this approach can be tedious and may not work if the base medium or cell line is altered during process development. A more optimal approach is to use a feed that can work across multiple base media and cells in a platform process.

B7 Determining Agitation Requirements for Microcarrier Processes: Method Development in the Mobius CellReady 50L Single-use Bioreactor Kara Der, EMD Millipore The Mobius® CellReady bioreactors have been demonstrated to provide a scalable platform for optimal suspension cell culture performance. However, not all cells can be grown in suspension. Adherent cells require a solid surface upon which to grow and have traditionally been grown using 2D tissue culture flasks or roller bottles in which scale-up becomes cumbersome. Alternatively, adherent cells can be grown in 3D suspension culture if a solid surface, such as a microcarrier, is provided. In order to insure successful cell culture, the microcarriers must be kept in suspension throughout the duration of the process. A methodology for determining the minimum agitation rate required to maintain adequate microcarrier suspension and thereby avoid microcarrier settling has been developed using collagen coated microcarriers (SoloHill) in the Mobius CellReady 50L bioreactor. We have demonstrated that by maintaining a constant agitation rate it is possible to maintain a similar suspension environment as the volume of the process is increased as long as the microcarrier concentration remains constant. Additionally, we have employed the Zwietering correlation to predict, and experimental analysis to verify, the agitation rates required to maintain suspension of different concentrations of microcarriers.

B8 Virus production in the Integrity® iCELLis® disposable fixed-bed bioreactor from bench scale to industrial scale Stephanie Dubois, Pall Life Sciences The iCELLis bioreactor from Pall LifeSciences is designed for adherent cell culture applications. Cells grow on macro-carriers packed in a fixed-bed, providing up to 500 m² of growth surface area in a small reactor volume. Controlled conditions, combined with the large growth surface area in the iCELLis yields high cell density and productivity. A number of viruses intended for use as vaccines, gene therapy vectors or oncolytics, including MVA, Bovine Herpes, Paramyxovirus, Influenza, Adenovirus, rAAV and more have been produced in iCELLis bioreactors. For initial feasibility studies, processes were tested using identical process parameters in benchtop iCELLis bioreactors as in standard static culture or roller bottle processes. HEK 293 cell cultivation was scaled up to an industrial scale 133 m² iCELLis bioreactor. MDBK and Vero cell cultures were scaled up to 66 and 660 m², respectively. Production of influenza, BHV and an undisclosed lytic virus was scaled up to 20, 66 and 660 m², respectively. For all viruses tested, results indicated that the iCELLis bioreactor is a suitable platform for industrial scale production.

B9 An Novel Method for Fermentation Process Optimization in Complex System Helena Fan, Effinity Tech Design of experiment (DoE) has been widely used for fermentation development and optimization. However, the commonly used DoE methods have some limitations and are not efficient to deal with very complex systems with 20 or even over 30 factors. A novel methodology, Directional Control Technology (DCT), based on system engineering approach, was used to optimize the base media, feed solutions and fermentation process all together as a whole solution for a newly constructed E. coli XS strain: total 66 parameters were included in the study. After a 2-phase study, 14 fermentation runs (treatments) were implemented in the 1.3 L DASGIP parallel microbial fermentor system. The optimized conditions, including the improved base media, feed solutions and optimized fermentation parameters, resulted in increase of yield of the target protein to 13.2g/L from 4.2g/L at the initial baseline conditions. The new methodology also suggested and quantified the top impact factors on the productivity of the target protein, including the fermentation operation parameters, such as pH, agitation speed, feed rate, DO, temperature; and the media component, such as Kanamycin, L-Alanine, Yeast Extract, etc, providing important information for controlling the system. Overall, with a few runs, the novel methodology enables to optimize the complex system with 66 parameters and achieved a high titer.

B10 Seg-Flow® Automated Nutrient Monitoring and Continuous Feed Platform for Precise Nutrient Feed Control Ashley Fisher, Flownamics, Inc. Fed-batch culture processes are commonly used for the production of various biotherapeutic and other industrial commodities. A widely employed nutrient feed control strategy for both cell culture and microbial fermentation processes is direct feedback control, in which the nutrient feed is directly controlled by the nutrient, i.e., glucose, concentration. However, various iterations of this feeding approach exist which may not be conducive to culture growth and productivity. In order to optimize culture performance, the means by which the substrate is fed to the culture is just as, if not more, important as maintaining the desired media substrate levels. An automated feedback control platform, which employs a continuous feed strategy based on the culture's real-time nutrient consumption rate, has been developed by Flownamics®. This integrated platform consists of the Seg-Flow® automated on-line sampling system, a nutrient analyzer and a feed pump. The Seg-Flow system was evaluated for its ability to precisely control media glucose concentrations in both low and high consumption rate culture simulation models. The results showed that the integrated Seg-Flow system was able to precisely control media glucose concentrations at the prescribed levels through real-time glucose consumption rate calculation and continuous feeding.

B11 TrafEn: A novel reagent that enhances polymer based gene delivery for use in biologics production Yee Meng Heng, A*STAR TrafEn is a breakthrough solution that enables efficient cationic polymer based transfection for viral production, rapid recombinant protein production and cell line development using mammalian systems. The industry is facing an immense pressure of having to develop cost effective systems for biologic production. Cationic polymers have gained popularity due to its ease of production, high scalability and affordability. Advances in the transfection technologies have been focusing on overcoming some of the key limitations such as low yield and quality. To address this crucial challenge, TrafEn was rationally designed to enhance the efficiency of cationic polymer based gene delivery by directing the trafficking of plasmid away from the acidic compartment and enhancing nuclear trafficking. TrafEn significantly increases the transfection efficiency of cationic polymers resulting in cultures containing a larger percentage of highly-expressing cells. High gene delivery efficiency is highly favorable and critical in both transient (TGE) and stable gene expression system (SGE).

B12 Efficient Strategies to Deliver Reliable and High Quality Biomanufacturing Processes Through Optimization of Cell Culture Media, Feeds and Process Parameters Timothy Hill, Fujifilm Diosynth Biotechnology Development of a reliable and high quality biomanufacturing process for recombinant CHO cell lines presents challenges from diverse nutritional requirements observed with different clonally derived cell lines, and variable response to process operating ranges. To address these challenges, we will discuss efficient approaches to optimize CHO cell culture media, feeds, and process parameters within FUJIFILM Diosynth Biotechnologies (FDB). Also discussed is FDB's new ApolloTM CHO expression system, a cornerstone of process scale-up efficiency offering robust gene expression in a platform media and feed production system. In this presentation, we outline efficient development strategies using informative case studies: (i) establishment of platform CHO cell culture media and feeds by implementing high-throughput media and feed development with DOE-based methodologies; (ii) demonstration of rapid optimization at bench scale including 2L/10L scale bioreactors; (iii) establishment of a "Toolbox" strategy targeting media, feeds, and process conditions to achieve desired mAb productivity and quality, with good process scale up demonstration.

B13 Case Studies of hERG Membrane Protein and Japanese Encephalitis Virus Production Using a New Innovative Moving Bed Bioreactor Lewis Ho, BioReactor Sciences Human ERG membrane protein has gained great interest as a target for drug discovery. A two fold increased expression of the hERG gene was induced under nutrient limitations. The protein is membrane bound and therefore whole cell recovery from the carriers is required. HEK293 cell was used. Japanese encephalitis virus (JEV) for vaccine has long been the only reliable solution to prevent deadly encephalitis. The virus however is unstable and post-infection conditions for optimal virus production and recovery is critical. We use two case studies to illustrate a new innovative Moving Bed (MB) bioreactor's unsurpassed versatility and functionality to accommodate specific desired conditions and to accomplish the entire upstream processes in one single bioreactor. The mobility of the moving matrix bed allows for the least amount of volume required for seeding, yielding high seed density resulting in increased cell attachment. Similarly, this feature results in high virus infection and DNA transfection efficiency. The MB system facilitates the principle similar to roller bottles intermittently exposing and submersing cells to air and medium to achieve maximum oxygenation with near minimal shear, and also eliminating foaming issues. The moving bed sets a new precedent in performing the mechanism of cell detachment and recovery in place.

B14 Particulates in Single-Use Bags Mike Johnson, Entegris The utilization of single-use systems in downstream applications is rapidly increasing. With this increase comes a heightened concern about particle contamination and the associated potential for health and safety risks. Particles found in single-use systems can be traced to several sources, including the system's materials of construction, its physical attributes and the environment in which the product is made. Particles found in drug products can have real patient implications which may include reduced drug efficacy or the blocking of blood vessels resulting in tissue or internal organ damage. The risk associated with particles depends upon several factors including the size, shape and quantity of particles present; the composition of the particles; the physical attributes of the patient; and the route of drug delivery. In order to better understand the impact of single-use systems on the particle content of drug products, we evaluated six different single-use bags from five different manufacturers, quantifying the number and size of particles present in each. This presentation provides the results of our evaluation, along with a comparison of the data to the limits established for USP <788> Particulate Matter in Injections.

B15 Aggressive Pichia, E. coli, and Bacillus Cultures Using the Thermo Scientific HyPerforma 30 Liter and 300 Liter Single-Use Fermentors Nephi Jones, Thermo Fisher Scientific Fermentation production facilities can utilize the Thermo Scientific™ single-use fermentor (S.U.F.) instead of traditional stainless steel cleaning in place/sterilization in place (CIP/SIP) fermentor vessels without modifying their existing procedures. As expression systems are moved to production volumes, they can easily be scaled from a benchtop fermentor (or flask) into the S.U.F. We demonstrate various cultivations exponential feed, high gas flow, seeding at 20% working volume, and comparison of the S.U.F. to traditional stainless steel fermentors. The HyPerforma S.U.F. is designed to be a fermentor and thus meets the performance requirements of dense, rapidly growing microbial cultures while offering the benefits of quick process setup, reduced contamination risk, and high production quality of the original Thermo Scientific™ HyPerforma™ Single-Use Bioreactor (S.U.B.).

B16 Enhancing cell culture process and overcoming challenges for antibodies utilized in site-specific conjugation Takuya Kikuchi, Agensys Possessing both the efficacy of cytotoxic drug and tumor selectivity, antibody-drug conjugates (ADCs) have been emerging as a new class of anticancer therapeutics. Traditional lysine- or cysteine-based conjugation technologies generate heterogeneous ADC molecules, which exhibit different pharmacology and present challenges to controlling safety, as well as manufacturing and analytical characterization. The site-specific ADC technology we are using produces ADCs with the same drug-antibody ratio. While homogenous ADCs are beneficial, in some cases a significant challenge associated with this technology is the low expression of antibody protein. In this work, we investigated how feed media components and cell culture processes affect protein expression and cell growth. In particular, we found that some feed media components strongly inhibited antibody production. By optimizing the feed medium and the cell culture process, we improved antibody titer by 75% compared to the original process.

B17 Perfusion CHO Cell Culture in BioBLU® 5p Packed-bed Single-use Vessel Nick Kohlstrom, Eppendorf The market for secreted recombinant protein therapeutics, including blockbuster drugs in the form of humanized monoclonal antibodies (hmAbs), has become a multi-billion dollar industry with the expectation of continued growth. One of the most cost effective methods for the production of secreted proteins is the packed-bed vessel operated under perfusion conditions. The maximum cell densities achieved in a packed-bed vessel is typically much higher than suspension cell culture or microcarrier-based adherent cell culture. The protein harvest can be carried out continuously, providing unparalleled product yield. This poster provides an example of using a BioBLU 5p packed-bed single-use vessel to conduct perfusion CHO cell culture producing a secreted hmAb. The BioBLU 5p vessel (pre-loaded with Fibra-Cel® disks) was controlled by a New Brunswick™ CelliGen® BLU benchtop bioreactor. The BioBLU 5p vessel was inoculated at an initial cell density of 0.3 x 10^6 cells/mL. Fourteen days of perfusion cell culture was conducted with a working volume of 3.75 L. Glucose, lactate and hmAb concentration were monitored daily. The glucose consumption rate was used to estimate the cell density in the packed-bed vessel. After 12 days, the culture reached to a peak cell density of approximately 10 x 10^6 cells/mL.

B18 Efficient Expansion of Suspension CHO Cells in Corning® New 5 Liter Erlenmeyer Flasks Anne Kwei, Corning Incorporated Erlenmeyer flasks are commonly used for expansion of a variety of suspension cell lines for bioprocessing applications. We have recently developed a 5L Erlenmeyer flask with the same footprint as a traditional 3L Erlenmeyer flask. The 5L shape has been optimized for increased gas exchange compared to the more traditional Erlenmeyer flask designs. Due to gas exchange limitations, most traditionally shaped Erlenmeyer flasks can only accommodate approximately one third of the stated volume of the vessel during culture. Our new 5L shape design allows for the culture volume to be increased to one half of the stated volume of the flask, resulting in a greater number of cells cultured in the same footprint as a 3L flask. In this study, we describe a protocol to scale up suspension CHO cells from 40 mL to 15L of total volume in just 12 days using our 5L Erlenmeyer flasks. Furthermore, we demonstrate that our new 5L Erlenmeyer flasks provide a more favorable culture environment (higher dissolved oxygen and lower lactate metabolite accumulation) and support a higher yield of viable CHO cells when compared to other commercial 5L Erlenmeyer flasks.

B19 Development and characterization of an orbitally shaken 12 L bioreactor suitable for mammalian and microcarrier cultures David Laidlaw, Kuhner Shaker Inc On the pharmaceutical market there is a demand for single-use bioreactor alternatives to rocking bags for culturing mammalian cells in the 2L-10L scales. Orbitally shaken vessels expose cells to low shear stress, making them suitable for the cultivation of shear sensitive organisms. In this work a disposable 12 L culture vessel was characterized. The vessel was constructively extended to a bioreactor with the integration of an online monitoring system (pH and DO). The influence of the integrated sensors and microcarriers on scale up parameters was investigated. This set of data presented will support all users of shaken bioreactors to optimize their specific cell cultivation conditions.

B20 Impact of raw material variability on glycosylation profile of a CHO-derived humanized monoclonal antibody Jayanthi Lakkyreddy, Genentech Variability and a shift in glycosylation profile were observed during process development for a CHO-derived humanized monoclonal antibody. This shift was attributed to the change in raw material lots, specifically ferrous sulfate (FeSO4). The trace metal manganese (Mn) can be an impurity in FeSO4, and is a co-factor for many of the enzymes involved in the glycosylation pathway. It was observed that some cell lines were more sensitive than others to minor changes in the concentration of trace elements such as iron. For such cell lines, the presence of trace element impurities in raw materials poses a risk for maintaining consistent product quality. Studies were performed to overcome impurity-driven variability in glycan profile by increasing the target Mn concentration in the production media. The results from these studies showed that the glycan distribution was more consistent when the Mn concentration was increased to levels above the impurity range coming from the FeSO4 raw material.

B21 A new DO control strategy of mimicking large scale DO heterogeneity to improve the cell culture scale down model Weimin Lin, Biogen Idec Oxidative stress is caused by elevated production or reduced detoxification of reactive oxygen species (ROS) in cells. ROS can directly damage the cells and is associated with cell death. When scaling up a process from bench scale 3-L bioreactors to 15000-L bioreactors, some cell lines were observed to have more than 50% growth decrease. The hypothesis is that the localized high DO exists due to mixing heterogeneity at large scale. To advance understand and control our bioprocess, we developed a scale-down model based on the impact of high DO using a new DO control strategy at small scale to mimic large scale DO heterogeneity, resulting in performance that is similar to large scale experience. The improvements to the cell culture scale down model and future work will be described.

B22 Effect of UV Treatment on Cell Culture Media Ted Mao, Trojan Technologies A serum-free, chemically defined media was irradiated with UVC at several UV Dose levels. UV Dose was verified using a challenge virus with measured UV inactivation kinetics. CHO cells were cultured in the media, and growth rates, viability and antibody productivity were assessed. Chemical composition of the media was also measured using quantitative NMR. Results showed that the UV treatment had no negative impact on cell growth or antibody production, even though some composition changes were seen.

B23 Real Time Monitoring of Various Growth Parameters in a Mammalian Cell System Daniel Mardirosian, Worcester Polytechnic Institute Monitoring cell culture processes is important for biomanufacturers and research investigators. Measuring in real time the growth parameters, nutrients and metabolites is critical for the productivity of the process. Timely and accurate data enables research scientists and students to better understand cell growth and the effect of the variable in the growth environment on cell yield and viability. This poster presents real time data collected utilizing The Cedex Cell Analysis System and Cedex Bio Bioprocess Analyzer during a series of experiments conducted to provide training for the scale-up a mammalian cell culture process at the Biomanufacturing Education and Training Center, WPI. These reliable technologies provide accurate data, demonstrating the utility of monitoring growth parameters for in-depth process understanding.

B24 Development of a High Cell Density Fed-batch Insect-cell Based Process Platform Joshua Merritt, MassBiologics of the University of Massachusetts Medical School MassBiologics is currently developing a high cell density fed-batch insect-cell based process platform for the production of proteins, virus-like-particles and viruses for use as human therapeutics. In order to achieve this end, the impact of feeding three different types of nutrient pools on Sf9 cell culture growth was tested using a design-of-experiments (DOE) approach. In the ranges tested, yeastolate and MassBiologics' chemically defined feed (a protein-free mixture of amino acids, vitamins, trace minerals and other cell culture nutrients) both individually and combinatorially increased maximum culture cell densities while a chemically defined lipids mixture had no significant impact. A yeastolate feed rate was identified above which the maximum cell density obtained was reduced, putatively, due to toxic metabolic by-product accumulation. Maximum cell densities increased with increasing feed rates of MassBiologics' chemically defined feed. Spent medium analysis indicated depletion of several amino acids during the shake flask screen. The feed medium was supplemented with these amino acids and modified feeds and feed rates based on the shake flask experiment were implemented in a 1 L scale bioreactor experiment. The highest cell densities obtained during this experiment were approximately 3-fold higher than those obtained in an unfed bioreactor culture: 35.9e6 cells/mL vs. 12.0e6 cells/mL. Rates of baculovirus infection at various cell densities were measured using a green fluorescent protein expression assay.

B25 Maximize without Compromise: Using super-concentrated feeds in fed-batch cell culture Becky Moore, Thermo Fisher Scientific High performance commercial Chinese Hamster Ovary (CHO) protein expression systems require fed-batch processes that maximize bioreactor working volume, maintain specific productivity (i.e., product per cell), and provide sufficient nutrient delivery to maintain proper protein quality. The challenge in media development is to overcome traditional solubility limitations that impacts initial bioreactor and feed volumes causing product dilution and compromised bioreactor yield. The development of this new technology enables simple, pH neutral, super-concentration of feeds up to 200g/L, which permits either reduced product dilution via feed volume or increased titers with additional feeding as desired. Recent studies using a CHO-K1 cell line demonstrated that after super-concentrating the feed by 2x, additional feed supplement could be added to the bioreactor resulting in a high performance fed-batch process exceeding 7g/L yield. This study highlighted manufacturing advantages including single-part water-only reconstitution, higher titers due to the use of this super-concentrated format, higher volume bioreactor inoculum, and flexibility in bioreactor usage. Incorporation of this technology into fed-batch bioprocesses enables flexibility in bioreactor start volumes and feed volumes, minimizes risk associated with multi-part extreme pH feed strategies, and allows for superior culture performance in recombinant biotherapeutic drug manufacturing.

B26 Development of Next Generation Feed Medium to Support High Performance CHO Cell Lines Brandy Nunez, Irvine Scientific There is an increasing need for concentrated media capable of supporting the nutrient requirements of modern, high-performing cell lines to keep pace with rapid advances in cell line engineering. This study highlights the development of a feed medium designed to maximize the yield of these modern cell lines while maintaining desirable product quality. Feed optimization utilizing DoE approaches allowed for targeted enrichment of limiting amino acids and critical components versus simply enriching the entire formulation. Nutrients in the newly developed feed were enriched by three-fold over the current generation, maintaining a conservative feed volume. This study will report the result of feed optimization, which produced an excess of 5 g/L of protein with peak cell densities over 25 million cells/mL, as well as its impact on product quality. The development of this next generation feed realized the potential of a high performance cell line.

B27 Gaining Optimized Cell-Level Observability and Data-Driven Process Guidance by Leveraging At-Line Tools such as the Modular Automated Sampling Technology (MAST^TM) Platform and Automated Sample Analysis with Automated Data Collection and Processing Systems. Clint Pepper, Capsugel In implementing process analytical technology (PAT), biopharmaceutical companies are continually striving to gain a fundamental understanding of what is happening to the cells within their bioreactors and the impact that the process has on the cell's ability to deliver the target product quality. While implementation of on-line tools like dielectric spectroscopy and Raman are helping to provide insight, there is still a gap integrating the data produced by these techniques with off-line measurements such as cell density, viability, metabolite levels, and titer and presenting that data to the end user that gives them real insight into their process. Bend Research Inc., in collaboration with Pfizer Inc. and other major biopharmaceutical companies, is working to advance the Modular Automated Sampling Technology (MASTTM) and automated data retrieval and analysis platforms, the goal of which is to provide a complete data management solution to the end user. The goal of the MAST and data processing platform prototypes is to cover all aspects of data management from providing aseptically collected bioreactor samples to analytical devices, maintaining the raw analytical data in a database and then providing the user with a Graphical User Interface (GUI) to rapidly modulate the data, providing instantaneous guidance. Early studies with the MAST platform have demonstrated that automated at-line measurements are representative of parallel manual samples while maintaining sterility. The data processing prototype effectively extracts data from MAST as well as from multiple analytical devices collating this data creating system observability and guidance for the end user. First this presentation describes how the MAST platform collects samples from bioreactors and initiates automated analysis via analytical equipment and second, how the data processing system retrieves the resultant data and pulls it together for the end user to deliver overall cell-level "observability." From this new level of insight into the operation of the system, scientists can obtain enhanced data-driven "guidance" for key activities like cell-line selection and optimized process operation. With this new technology, the bioprocess industry can make major advances toward advanced real-time testing, predictive control, and overall enhanced bioprocess design and operation.

B28 Optimization of the CHO host and expression technologies to reduce timelines and improve productivity and quality Holly Prentice, ExcellGene SA Chinese hamster ovary cells (CHO) have been the preferred host for biomanufacturing of complex therapeutic glycoproteins for more than two decades. Conventional approaches with these cells for the development of a manufacturing system have inherent limitations in time required, productivity and quality. In order to reduce these limitations optimized host cells, expression systems, and innovative procedures should be employed. An optimized host cell line, CHOExpress™, which has a doubling time of less than 14 hours and can achieve cell densities of 14E6/mL in batch culture has been developed. In addition, a unique transposon based expression technology capable of generating >200 mg/L at the pool stage has been integrated with the CHOExpress™ host. These two technologies, in concert with high-throughput process optimization approaches, have been used to generate stable cell lines in 12 weeks and high-yield processes (multigram/L) including stability assessment in 8 months.

B29 Inclusion of a Micro bioreactor system in the MaxXpress platform for Cell Line & Process Development Jason Rahal, Cobra Biologics ltd Cobra Biologics is an established manufacturer of monoclonal antibodies, other recombinant proteins, DNA and virus. Over the last 15 years we have supplied more than 320 batches to more than 100 customers located in 19 different countries on all continents of the world. The need for rapid cell line and process development push processes to higher throughput and smaller scale. Tight time lines and development costs are crucial when developing a platform approach for antibody production, from cell line development to GMP manufacture. Evaluation of cell culture media and additives, as well as process parameter optimisation, can be very time consuming. An automated, disposable micro bioreactor system was included in Cobra's maxXpress platform. This was to provide an approach where cell culture media and additives can be tested while maintaining bioreactor process parameter control, thus facilitating subsequent scale up of the fermentation process.

B30 MANUFACTURING OF A CHO-DERIVED MONOCLONAL ANTIBODY - A SCALE-DOWN APPROACH FOR A SENSITIVE CELL LINE THROUGH BIOREACTOR DESIGN AND CONTROL PARAMETER MODIFICATIONS Delyan Rusev, Gilead Sciences Scale translation of monoclonal antibody (mAb) manufacturing processes often presents an operational challenge due to the fact that many volume-dependent parameters scale non-linearly, and cannot be matched across bioreactor volumes. For this reason, small scale or pilot scale performance may not translate directly to large scale bioreactors, resulting in lower productivity and differences in growth and metabolite profiles. For a monoclonal antibody process using a sensitive CHO cell line, differences in growth, metabolic, and control profiles were identified between manufacturing scale and the small scale model originally used to develop the process. Various bench top bioreactor modifications (configuration and control strategies) were evaluated in order to mimic the large scale performance. A final scale-down model addressing multiple hydrodynamic shear parameters, such as bubble size, mixing rate and bubble distribution was developed to effectively reproduce selected performance attributes of the large scale production culture. The scale-down model was then used to assess potential process improvements that would optimize the performance of the large scale bioreactors.

B31 Efficient expansion of human mesenchymal stem cells (hMSCs) on Corning® Enhanced Attachment microcarriers using a continuous agitation protocol Rachel Ryznal, Corning, Inc. Microcarriers are small spheres used to culture adherent cells at scales impractical using traditional two-dimensional (2D) cultureware. Although 2D culture methods are well established for the majority of cell types, protocol optimization is required for microcarrier-based cell culture. Corning developed and recently commercialized several gamma sterilized, ready to use microcarriers with different surface chemistries for different cell types. In our earlier studies we reported efficient expansion of hMSCs on Corning® Enhanced Attachment (EA) microcarriers using an intermittent agitation protocol with an initial static attachment phase. Here we report a continuous agitation protocol for hMSC expansion on EA microcarriers in 1L glass spinner flasks. The following conditions were evaluated: cell seeding density, microcarrier surface type, microcarrier concentration, agitation rate, working culture volume, and +/- serum during attachment. We achieved consistent hMSC expansion across multiple cell donors to 50,000 cells per cm2 (200,000 cells/mL) with minimal microcarrier clumping (2-3 beads/clump). Higher cell densities could be attained but with larger clump sizes (>5 beads/clump). After culture on microcarriers, cells retained typical spindle-like morphology, cell surface marker expression profile, and normal karyotype. The key learnings from these optimizations are being applied to bench-top scale stirred tank bioreactors.

B32 Efficient Expansion of Vero Cells on Corning Microcarriers and Demonstration of Scalability in a Benchtop Bioreactor Jeffery Scibek, Corning Incorporated Biopharmaceuticals currently represent the fastest growing segment of the pharmaceutical industry. The increased interest in biopharmaceuticals is fueling the demand for more efficient and innovative cell culture products. To address the need for increased cell yields, Corning has recently commercialized a panel of gamma sterilized, ready to use microcarriers for the high yield culture of anchorage-dependent cell lines. Here we report a protocol for expansion of Vero cells on Corning Enhanced Attachment microcarriers for vaccine applications. The following culture conditions were optimized in 1L Glass spinner flasks to achieve an efficient cell attachment (>90%) and expansion (>10 fold): microcarrier surface, microcarrier concentration, cell seeding density, cell seeding volume and agitation speed. With this optimized protocol, we routinely achieved >90% attachment efficiency within 2 hours and more than 2,000,000 cells/ml (200,000 cells/cm2) cell yield, which corresponds to ~13 fold cell expansion in 5 days. The scalability of this protocol to a 5L benchtop stirred tank bioreactor was successfully demonstrated. In summary, we developed a protocol for efficient expansion of Vero cells on Corning gamma sterilized, ready to use microcarriers. This system provides an attractive method for large-scale expansion of Vero cells for bioprocessing applications.

B33 Development of Serum Free Complete Medium for BHK21 Neelanjan Sengupta, Becton Dickinson BHK-21 is an industrially relevant cell line that is used for production of various viral vaccines associated with animal health, such as foot and mouth disease (FMD), Japanese encephalitis, Rabies, and Aujeszky's disease. BHK-21 cells are often grown in basal media supplemented with 10% serum. Aside from the cost of serum itself, serum qualification processes are time-consuming and expensive but necessary to control lot-to-lot variability. Peptones, which are hydrolysates of plant or animal origin, are known to enhance various biopharmaceutical production processes. In addition to cost reduction, peptones also help in retaining physiological properties, such as binding and neutralizing toxic components; maintaining buffering capacity and provide nutritional factors, growth factors and shear protection for the cells. Here we describe a process for the rapid development of a peptone based serum-free complete medium. The process entailed a preliminary screening of the complete media formulations containing BD peptones in a reduced serum condition. This was followed by achievement of serum-free conditions by resolving the issue of cell clumping. Finally, cell growth was further enhanced through optimization of the medium. The approach highlighted, resulted in an easy to use serum-free complete medium that eliminated the need for serum supplementation for BHK-21 cells. The BHK-21 cells could be adapted directly from 10% serum media to BD serum-free medium without the need for multiple weaning steps.

B34 Evaluation of the BioFlo® 320 process capabilities Ma Sha, Eppendorf Inc. Historically, stirred-tank fermentors and bioreactors have been the trusted design for cultivating all types of submerged cultures including suspension and anchorage-dependent mammalian, insect, yeast, plant, and microbial cultures. The tried and tested tank design offers scalability and proven reproducibility which is pivotal for cost-saving process development and productivity. However, for the most part, the glass or stainless steel autoclavable systems and the single-use equipment have remained separate. In order to meet the demand of improved process flexibility and cost savings, Eppendorf recently developed a new bioprocess controller that combines the capabilities of autoclavable and single-use systems into one advanced bioprocess control station, the BioFlo 320. The newest offering in the Eppendorf bioprocess portfolio, the BioFlo 320 blends design and utility in one all-inclusive package. An industrial controller design, interchangeability of autoclavable and single-use vessels, intelligent sensors, Ethernet connectivity, and an enhanced software package are only a few of the features that set it apart from the competition. This poster characterizes the BioFlo 320's capabilities in terms of key process parameters including oxygen transfer rate (OTR), volumetric mass transfer coefficient (kLa), water and energy consumption, tip speed, specific power input, and Reynolds number. These data allow a quantitative analysis of bioprocess engineering parameters, thus allowing potential users to gain insight into the system's high density cell culture and fermentation capabilities.

B35 Differential Effects of CD and Peptone Media Formulations on Cell Growth, Protein Production, and Protein Quality Seemab Shaikh, BD Biosciences The quality of recombinant protein produced by a cell line is a critical aspect of evaluating a cell line's performance in a biopharmaceutical process. The cell culture medium in which cells are grown can have a significant impact upon protein quality, independent of the effects on growth and protein production. Therefore, it is necessary to assess protein quality during the process of cell line development and media selection. We have demonstrated that changing the culture medium, as well as single components within a medium, can have a notable impact on protein quality. Integration of key components into the base medium, as well as feeding specific components, were two strategies used to improve protein quality and biological performance of the CHO cell line tested.

B36 Alternative Splicing and Method for Specific Detection of Splicing Variants in CHO-K1 Cell-line during Fed-batch Production Process Zhixin Shao, Roche Diagnostics Alternative splicing is a process by which the exons of the RNA produced by transcription of a gene are joined in multiple ways during RNA splicing. The resulting different mRNAs are usually translated into different protein isoforms. In this way, a single gene may code for multiple proteins. Amyloid precursor protein (APP) is an integral membrane protein expressed in different cells/tissues in many organisms. It has been previously reported that Chinese hamster ovary (CHO) cells express full length APP. The biological function of APP in CHO cell-line remains unclear. Currently no detailed information about CHO APP gene structure/expression in K1 cell-line is yet available. In addition, no alternative splicing isoforms of APP have previously been detected in CHO cells. In this study, novel splicing test methods based on amplifying and sequencing APP mRNA isolated from cell samples have been developed. Described is the identification of at least four (4) different splicing variants of the APP mRNA in CHO-K1 cells. These mRNA variants occur mainly through skipping one or more full exons within the APP gene. These methods allow for improved detection limits of spliced RNA variants in cell samples particularly from a fed-batch production process.

B37 VAYU meter - Bio monitoring device Moria Shimoni, VAYU sense VAYU sense's rapid screening device, the "VAYU meter", has been developed for the detection of living cells and microorganism's in growth medium. The VAYU meter measures CO2 metabolic gas concentrations produced during cell's respiration and proliferation. VAYU meter enables continuous metabolic gas detection for high sensitivity bio-process control, monitoring the growth rate and bio concentration. By means of IR absorption, our device allows in-situ detection of cells and microorganisms in fermentors or bioreactors directly throughout the reactor exhaust without the need for opening, sampling or incubating. This device can be applied for quantitative analysis and estimation of a level of biological activity of cells and microorganisms in pharm processes. Fermentation processes of most important and complex pharmaceuticals require precise culture base growth. From the early stages of commercial proteins production, the handling of cultures is being subject to challenges. Commercial production of products on a large scale depends largely on the stable maintenance of cell level. To optimize biotech production processes and improve yield and economics there is a need to achieve better, precise, real time process understanding and improved equipment utilization. The VAYU meter is a real-time process status monitoring solution capable of accurately indicating relative reaction conditions. This high resolution continuous detection system is successful in detecting cells and microorganisms' concentration. VAYU meter - Advantages and Features ? Enables to control culture growth throughout all stages of the fermentation process ? Contact-less and easy to install on every system (fermentors; bio-reactors, etc.) ? Real-time monitoring of biomass production process ? Shortens R&D stages ? Optimized production processes ? Improved yield

B38 Large-scale Production of Human Mesenchymal Stem Cells in BioBLU® 5c Single-use Vessels Khandaker Siddiquee, Eppendorf, Inc. Stem cell-based regenerative medicine has great potential to revolutionize human disease treatments. Among the various stem cell platforms, mesenchymal stem cells (MSCs) represent one of the highest potentials as evidenced by clinical trial activities. Currently, there are over 400 clinical trials based on MSCs registered at clinicaltrials.gov. Although successful expansion of MSCs in vitro has been well established, the large clinical-scale production of MSCs remains a bottleneck, potentially limiting the immediate clinical applications should some of the ongoing trials receive FDA approval. In this study, we demonstrate the success of large clinical-scale culture of human adipose-derived mesenchymal stem cells (AdMSCs) in an industrial single-use vessel at 3.75 liter (L) scale (working volume). The vessel offers a precision controlled environment for the ideal growth of stem cells under simulated physiological conditions. Stem cells and culture media were monitored, analyzed, and controlled, thus allowing us to produce AdMSCs in large clinical-scale quantities while maintaining healthy stem cell properties as evidenced by stem cell marker assays and differentiation assays performed at the end of the culture. Furthermore, with clinical relevance in mind, every cell culture step from T-flask to shake flask to bioreactor vessel was conducted strictly using single-use consumables.

B39 Improving solubility, homogeneity and convenience of dry powder cell culture media by physical modification Nikolai Stankiewicz, Merck KGaA, Germany, EMD Millipore To leverage the advantages of dry powder cell culture media (CCM) with respect to shipping and storage they need to be highly soluble, homogeneous and convenient to handle. An improvement of dry powder media properties can be achieved by the combination of optimized milling and suitable formulation technologies. Starting from homogeneous dry powder CCM the roller-compactation technology presented in this poster fixes the homogeneity in larger compactate particles preventing any de-mixing. Aside the presented roller-compactation offers several other advantages based on the fact that only pressure and no water is needed in the production process leading to a reduced water content of the product. To prove that this physical processing of the raw materials did not derogate the integrity of sensitive CCM components these were monitored by HPLC and LC-MS. Applying media as compactates also increases the flowability making the handling of these CCM much more convenient. But the key advancement of using compactated media is the acceleration of the dissolution speed. The increase in dissolution speed was analyzed by on-line conductivity and particle size distribution measurements showing a 50% reduction of dissolution time. Finally the applicability of compactated CCM is proven with fed-batch cultivation experiments based on compactated CCM.

B40 An Integrated Microbial Expression Toolbox to Enable QbD Process Development April Stanley, Cytovance Biologics, Inc. The Gram negative bacterium, E. coli, is a true workhorse in the biopharmaceutical industry and has a well-established track record of FDA approval and commercialization. Despite its broad utility at the bench, many commercial expression systems and hosts pose risks and obstacles in terms of cost of goods, scale-up challenges and in-process control. Researchers often lack the manufacturing expertise or understanding of E. coli genetics to design out the problems before they arise. Cytovance has developed a new microbial expression toolbox that works for manufacturing. Our integrated approach from DNA to DOE will be presented along with a case study.

B41 Continuous Bioprocessing and Continuous Biomanufacturing Abstract Title: Continuous Monitoring of Biochemicals Prashant Tathireddy, University of Utah A novel sensor system is developed for continuous monitoring of biochemicals in bioreactors. This system uses simple yet robust patented technology based on smart polymers and magnetic transduction mechanism. Here we present our latest experimental results on sensitivity of the sensors, affect of sterilization processes and selectivity. Current sensing methods use amperometric and enzymatic biosensors which suffer from small finite life-times, limited to few sterilization techniques and respond to small range of analytes. We have successfully demonstrated the efficacy of our technology by monitoring glucose, pH and osmolality of commercial media solution over the ranges of 2-50mM glucose, 5.5-8.5 pH, and 300-600 mOsm/Kg with high resolution. All the materials that come in contact with the media were successfully tested to be compliant with standard bioprocess sterilization techniques including gamma sterilization. We have seen no adverse affects due to common sterilization techniques. This was confirmed by comparison of sensor sensitivity and response time before and after gamma sterilization process. We also tested our sensors for selectivity by introducing possible contaminants and have seen minimal adverse effects. The sensor prototypes were integrated with Bluetooth 4.0 for data telemetry to a custom app on iOS devices. The technology presented here provides an inexpensive method of producing biosensors with unparalleled advantages. Smart polymer based sensors can be used for continuous monitoring with fast response times to a wide range of biochemicals. This technology also has a potential to sense multiple analytes using different polymers on a single platform

B42 Gas Analysis Mass Spectrometry Applications in Fermentation and Cell Culture Processes Pete Traynor, Thermo Fisher Scientific As a PAT tool, on-line analysis of the inlet and outlet gases of sparged fermentors, provides an ideal means of characterizing the fermentation state. Key indicators include the Carbon Dioxide Evolution Rate, Oxygen Uptake Rate and the ratio of these two parameters: the Respiratory Quotient (RQ). These measurements are used to monitor and control the process: for example to decide when to transfer a seed tank culture to inoculate the fermentor, to control sparging gas flow rate and composition, to detect contamination by foreign microorganisms and to control the substrate feed introduction rate. They are also used to determine the cell density, to compare with historical trend data to verify the process is proceeding normally, and to determine the completion of the batch. Process Mass Spectrometry is the most powerful technique for gas analysis of fermentations. The multi-component measurements are fast and precise. It has a wide dynamic range to allow measurements over the range of ppm to percent and also the sensitivity, stability and resolution to determine minute changes in concentrations. A mass spectrometer for fermentation is usually supplied with a multi-point sampler so that multiple samples are measured with one instrument. As an example, with typically 10 seconds being required per sample point, 60 sample points can all be measured with a cycle time of 10 minutes on one instrument. Additionally Process Mass Spectrometry has the flexibility to be able to monitor many different components. This poster describes the technology and benefits of fermentation monitoring by Process Mass Spectrometry.

B43 Scale Down Models for Upstream Cell Culture Processes: Maximizing Opportunities for Success in Technology Transfer and Process Characterization Eric Wallenstein, Merck & Co., Inc Scale-down models are commonly used to assess the impact of process parameters on critical quality attributes (CQAs) and key performance attributes (KPAs). Scale-down models are valuable in aspects of technology transfer and process characterization. In conjunction with technology transfer, the model is qualified at center point conditions to demonstrate that it is representative of large-scale process performance. The model can subsequently be used for process range finding studies (e.g., DoE or OFAT studies) and to support parameter range setting [e.g., proven acceptable ranges (PAR)] and ultimately regulatory filings. In addition, the model can be used for deviation investigations and to troubleshoot differences in cell culture performance observed in large-scale batches. Specific examples explored in this poster include assessing the effects of shear stress to the cells and dissolved oxygen levels on cell culture performance and product quality. In the latter example, the robustness of the scale-down model was suitability challenged and optimized to predict large-scale performance.

B44 High Throughput Screening for Mammalian Cell Clones Wen Wang, Massachusetts Institute of Technology For the past decade, biopharmaceutical industry had a fast growth, creating a hundred billion dollar market worldwide with the approval of recombinant proteins, monoclonal antibodies and nucleic acid–based drugs. Among all these production systems, the Chinese Hamster Ovary (CHO) cells are the most widely used mammalian cell systems for transfection, expression, and large-scale recombinant protein production, due to its advantages in accurate glycosylation for production of natural proteins in vitro. In recent CHO cell culture technology, significant improvement in recombinant protein production leading to a titer up to 10 g/L was achieved, to meet the high market demand by using stable and high producer together with the optimization of culture process. However, rapid selection for high production of clones is still needed. In current study, we apply a new screening concept for on line and in situ high-throughput screening for mammalian cell system. CHO cell line producing anti-her2 was used as the model mammalian cell system. By measuring the key parameters of the cell culture system on line, the performance of cell line can be predicted, and the high producers can be identified without performing invasive assays. The impact of the research will be very significant in upstream bioprocesses. In cell line development, product titer is a crucial parameter for clone selection. Our method does not require any external reporter, and it is a non-invasive liquid-based assay. We only need to monitor basic information of cell culture in order to calculate the product quantity. High and low producers can be easily identified in a high-throughput manner on-line and in situ using automated handling and aforehand programming. This will be a revolutionary change to current mammalian cell screening system. Keywords CHO Cells, High Throughput, Fast Screening High Producer, Product Prediction

B45 Effects of Cell Density, Aging and Media Formulation on CHO Antibody Expression: Feasibility of Titers >1 g/L. Weili Wang, MaxCyte, Inc Flow electroporation provides a means of fully scalable, highly efficient transient gene expression (TGE) that results in antibody titers >1 g/L and production of multiple grams of antibody from non-engineered CHO-S cells. Pre-transfection cell health, growth, and passage number can affect transfection efficiency and cell viability levels and, thus, antibody production. In this poster, data using flow electroporation via the MaxCyte STX® Scalable Transfection System are presented that examine the effects of pre-transfection parameters such as, media supplementation, cell density, and cell passage number on CHO-based antibody production. In conjunction, data illustrating the effects of post-transfection parameters such as seed density, media formation, and feed strategy are shown. Taken together these data will demonstrate the ability of MaxCyte STX electroporation to i) produce secreted antibody titers >1 g/L yielding multiple grams of antibody within two weeks of a single CHO transfection, ii) scale-up without protocol reoptimization while maintaining transfection performance, and iii) enable fine-tuning of pre- and post-transfection parameters to balance speed, cost, and antibody yield. In summary, these data will support the use of flow electroporation via the MaxCyte STX as an enabling technology that ultimately reduces early-stage labor and cost expenditures, while delaying the migration to stable cell line generation by extending the use of CHO-based TGE through mid- to late-stage antibody development.

B46 Development and characterization of large scale production of a recombinant rabbit monoclonal IgG Bill Zeck, Abbott Development of a robust immune response to small hapten immunogens is relatively difficult to obtain in mice compared to rabbits. We immunized rabbits with a vitamin-D immunogen and isolated six rabbit monoclonal antibodies directed against 25-OH vitamin D. All monoclonals exhibited similar binding characteristics despite divergent sequences within their respective complimentarity determining regions (CDRs). Cell culture process development and downstream purification processes were developed to ensure high levels of production of the recombinant IgG from a stable CHO cell line. Potency testing of three large scale pilot production runs revealed equivalent performance thereby demonstrating the robustness of the processes and their readiness for full factory implementation.

Formulations/Delivery

C1 Highly-Automated Procedures for the Assessment of Protein Formulations Russell Burge, Freeslate A highly automated process was devised to prepare and analyze samples for the formulation development of a stable protein. Automation increased efficiency of the activities compared to current methods and procedures. Forty-eight protein formulations were generated together with placebos. Automated procedures were employed for appearance (color, turbidity, and particles), viscosity, and pH testing. Spectrophotometry, chromatography, and light scattering were performed using semi-automated procedures. Performance of the formulations was evaluated after subjecting the vials to stress conditions.

C2 QbD Guided Process Development of a Novel Cationic Nanoemulsion for Delivery of Nucleic Acids Sonia Gregory, Novartis Vaccines Emulsions have been used to enhance potency of a vaccine or in some cases for delivery of an antigen. Here we discuss the development of a novel Cationic Nanoemulsion (CNE) for delivery of nucleic acid-based vaccines. CNE has been shown to be capable of safe and efficacious delivery of a 9 kb self-amplifying mRNA in small animal and non-human primate studies(1). CNE was developed based on the Novartis proprietary MF59 oil-in-water emulsion adjuvant. The development and scaled up production of CNE was guided by Quality by Design principles. The critical quality attributes were assigned to the product and assessed during each step of the process to define criticality of process parameters. Upon identification of critical process parameters, the studies to better define operating ranges were executed. Overall the use of QbD approach has eliminated redundancy and improved efficiency during the development of CNE process. (1.) Brito, L.A. et al. A Cationic Nanoemulsion for the Delivery of Next generation RNA Vaccines. Molec. Ther. 19 Aug 2014 10.1038/mt.2014.133

C3 Characterization of Protein Aggregates and Sub-visible Particles in Therapeutic Protein Formulations using ImageStreamx Imaging Flow Cytometry Christine Probst, EMD Millipore Protein aggregates and other types of sub-visible particles present within therapeutic protein formulations may impact drug safety and efficacy. USP <788> guidelines define limits for particles > 10 µm, however characterization of particles 2-10 µm is increasingly expected based on their potential immunogenicity. Accepted industry techniques light obscuration and flow microscopy provide particle size and concentration information, but offer limited ability to differentiate among the different types of contaminants routinely found in therapeutic formulations. ImageStreamx imaging flow cytometry is a potentially attractive platform for analysis of sub-visible particles based on its ability to rapidly collect robust, single-particle data sets consisting of multispectral brightfield, 90° side-scatter, and fluorescent imagery. Herein we evaluate ImageStreamx as a novel methodology for identification and characterization of sub-visible particles. Three common contaminant species- protein aggregates, silicone oil droplets, and bacteria- were fluorescently labelled and used as a model system. Particle data was collected on ImageStreamx and current industry standards HIAC and Micro-Flow Imaging. The results demonstrate ImageStreamx provides more sensitive detection of aggregates and facilitates identification of particles and their complexes based using fluorescent tracers. ImageStreamx should prove instrumental for developing processes that minimize drug degradation and understanding the role of particles on patient health.

C4 A New Approach to Protein Aggregation/Particle Characterization Data Analysis David Thomas, Lumetics Inc. Purpose:~~A wide range of analysis technologies are regularly applied to protein formulations for characterization, process/product optimization, and ongoing monitoring/control. Each analysis technique has its own operating principal, vendor-specific implementation plus range of instrument models and data reporting formats. Transcribing, aggregating, and visualizing large volumes of orthogonal and complimentary data is often time-consuming, repetitive, and inconsistently applied. This paper summarizes work done to leverage advancements in the data management/visualization field, and apply them towards the analysis of protein formulations.~~Methods:~~Parsing scripts were written to automatically bin and transcribe all measurement data plus analysis configuration info directly from equipment output files into a centralized database. A user-interface was constructed to allow inclusion of additional sample/process information (e.g. formulation/process attributes, experimental conditions, lot/batch information). Finally, a client-side data visualization interface was developed that would allow rapid construction and application of analysis templates (charts/tables/statistics) upon any subset of data within the database.~~Results:~~The data management and analysis architecture was applied to a selection of particle and protein aggregation characterization datasets generated using the following technologies: Flow Microscopy (MFI, FlowCAM), Light Obscuration (HIAC, Pamas), RMM (Archimedes), NTA (Nanosight), and Coulter (MultiSizer). The following analysis templates were created and applied to these data sets:~~a) Technology comparisons (count, size, morphology, and images)~~b) Sub-population development (morphological parameter filters, density filters)~~c) Process/product optimization (formulation and process attributes)~~d) Method development/qualification (analysis configuration optimization, ICHQ2)~~e) Forced degradation/stability (time, temperature, stress condition)~~f) Comparability studies/long term trends (product and process comparisons)~~Conclusions:~~A tool has been developed that will automatically bin and transcribe measurement data from analysis equipment into a centralized database, aggregate data according to specified database field filters, and present the data utilizing common charting, table, and statistical analysis elements. Use of the tool was found to eliminate transcription errors, save weeks of time manipulating data sets, and improve analytical insight and reporting consistency.

Manufacturing

D1 The Quincy College Biotechnology and compliance training program Isso Bayala, Quincy College With strong industry support, Quincy College has developed a Biotechnology and Compliance Program for training in bio-pharmaceutical manufacturing. Students are trained on both single-use (disposable) and traditional stainless steel bioreactor systems while following Good Manufacturing Practices (GMP) in a fully equipped, state-of-the-art facility. The program prepares students for entry level positions in biomanufacturing, quality control, quality assurance, and biomedical research. Short courses in numerous areas of biomanufacturing are offered for incumbent worker training. In addition to hands-on laboratory courses, students train on a highly interactive, comprehensive web based e-learning environment to practice industrial scale single-use biopharmaceutical manufacturing. Employment rate for students who have successfully completed the program is ninety percent.

D2 Accelerated Production of Therapeutic Monoclonal Antibodies by Continuous Biomanufacturing Maurizio Cattaneo, BioVolutions Inc Described herein is a rapid continuous biomanufacturing platform that combines a perfusion mammalian cell culture with a synchronized purification of the therapeutic antibodies produced by the cell culture without the use of intermediate holding tanks or other large media retention devices. The method described herein includes continuous cell culture of mammalian cells expressing the antibody of interest and comprising a cell retention device wherein the perfusion of fresh media into the reactor and hence the harvest rate of antibody containing spent media from the reactor has a rate of 1 vvd or less as a result of glucose control with a cell density of between 4x107 cells/mL and 6x107 cells/mL. Immediate recovery and purification of the antibody is obtained by synchronizing the rate of perfusion with the antibody capture and elution cycle of the affinity column containing between 0.1% 5% of the volume of the bioreactor. The perfusion and affinity capture steps were perfectly synchronized thus resulting in an order of magnitude increase in the antibody production rate compared to conventional fed-batch.

D3 Sensitivity Analysis of BioGTL Process Qiang Fei, National Bioenergy Center In light of the relatively low price of natural gas and increasing demands of liquid transportation fuels, attention has begun to turn to novel biocatalyst for conversion of natural gas into diesel fuel (Bio-GTL). A techno-economic analysis (TEA) was performed for an integrated process using fermentation process converting natural gas to microbial lipids and catalytic upgrading lipids to diesel blend stocks. A sensitivity analysis was carried out based upon the identified key parameters, such as plant capacity, process technology (yields and rates), productivity, and raw material cost. The sensitivity analysis cases were also presented to compare the production of natural gas-derived diesel with different fuels produced from various platforms.

D4 Successful Implementation of Automation in Single-Use Bioprocessing Todd Kapp, Parker domnick hunter Single-use bioprocess technology has become near ubiquitous within biologics manufacturing facilities. Benefits such as the avoidance of product cross contamination, flexibility process configurations and reduced capital expenditure requirements are especially attractive to contract manufacturing organizations. Single-use processing steps can be manual, semi-automated or fully automated. Many biomanufacturers are still evaluating the level of automation that is appropriate within their operations before making facility investments they will need to live with for years to come. This presentation will look at a project to automate a single-use, final bulk filtration and dispense processing step at Fujifilm Diosynth Biotechnologies, a biopharmaceutical contract manufacturing organization. A key component of the project was to define the level of automation that was appropriate for Fujifilm Diosynth Biotechnologies in order to deliver the required benefits of process control and consistency, increased speed and operational flexibility.

D5 The Use of Mobius MIX Vessels with Protein Solutions: Impact of Impeller Flow and Particle Shedding on Protein Turbidity and Aggregation Dana Kinzlmaier, EMD Millipore The increasing implementation of single-use equipment and assemblies in biopharm processes has resulted in more flexible facilities with faster batch-to-batch and product-to-product turnaround times. However, it has also resulted in new questions about how the materials of construction and system functionality might impact the drug products being produced. Customers considering a move to single-use mixing systems for buffer preparation have typically wanted information about particulate count and generation and extractables/leachables identity and levels as well as guidance on an effective mix protocol to ensure robust dissolution of buffer components. But when considering these same mixing systems for protein pools, additional concerns are raised about the potential impact of the impeller action and the particulates on protein quality and stability. To have a better understanding of the use of the Mobius MIX family for protein pools, we performed a study to determine whether we could detect a change in protein quality during mixing and whether the change could be correlated to a mixing parameter. In addition, we evaluated whether the particulates that are generated have an impact on short-term protein stability.

D6 High cell density fermentation of Escherichia coli using the New Brunswick™ BioFlo® 115 Benchtop Fermentor Bin Li, Enppendorf, Inc Escherichia coli (E. coli) is a Gram-negative bacterium that has had a long history in the world of laboratory and industrial processes due to its ease of manipulation and well understood genome. It is widely cultured under aerobic conditions. High cell density cultivation of E. coli is a powerful technique for the production of recombinant proteins. Indeed, 30 % of the FDA-approved biopharmaceuticals on the market are produced in E. coli. The New Brunswick BioFlo 115 from Eppendorf is an easy-to-use benchtop bioprocess system, with built-in controls for operation as a microbial fermentor. Featuring Rushton impellers, high speed direct drive motor, and optional thermal mass flow controller (TMFC) and automatic gas mixing, this system is ideal for use in R&D labs, universities, teaching facilities, testing labs and more. This poster presents a successful example of high density fermentation of E. coli using a BioFlo 115 fermentor with 2 L working volume heat-blanketed glass vessel. E. coli cultivation achieved a high optical density (OD600) value of 140 at 11 h without optimized fermentation medium and conditions.

D7 Industrialization of a cell based therapy process - How to identify the right technological strategy Fabien Moncaubeig, Pall Life Sciences Cell-based therapies are gearing up to have extensive impact on the healthcare field. This has set the stage for high productivity manufacturing technology with an emphasis on adherence to GMP standards while controlling cost of goods. Scale-up is not as simple as providing a larger surface area. Changing the niche environment has a big impact on cell behavior. The scale-up strategies from R&D to industrial production must preserve the integrity and quality of the cells. The presentation will cover the technological options and related challenges that are currently available. Particular focus will be made on the impact on the time-to-market.

D8 Microleak Detection in Flexible Bulk Bioprocess Containers and It's Correlation to Microbial Ingress Probability Vishwas Pethe, Pall Life Sciences The rapid adoption of single-use bioprocessing containers continues to raise persistent concerns about disposable system integrity. The paper discusses inadequacies of the existing integrity testing methods for flexible containers and presents two novel technologies for detecting defects in single-use containers at the container manufacturer and/or end customer facility. A controlled microbial ingress study conducted on flexible sterile containers revealed that defects as small as 12 micron are capable of causing sterility breach in aerosol challenge test while defects as small as 2.6 µm are causing sterility breach in immersion challenge test. A combination of helium and electro-detection allows testing of single-use containers before, during, and after use. Data will be presented demonstrating detection of 10 micron defects by helium method and 0.58 micron defects by electrical method.

D9 Innovative Pre-Packed Process Columns for Biomanufacturing Lynn Sutherland, Atoll Bio Greater adoption of single-use and disposable systems in biomanufacturing and development will require improved downstream device innovation, particularly for pre-packed chromatography columns. Atoll developed and evaluated a disposable, pre-packed, pre-qualified column platform, MediaScout® MaxiChromAC. MaxiChromAC is suited for the purification of biopharmaceutical compounds such as mAbs and vaccines in preclinical, clinical phase I and II studies. The column can also be used in full scale manufacturing depending on the scale and titer of the fermentation process. The innovative design of the column allows direct connection to state-of-the-art disposable chromatography systems. It also allows the packing of a broad range of commercially available chromatography media of different particle sizes including rigid, semi-rigid and soft polymeric backbones. The transparent column housing allows top to bottom bed inspection during the chromatographic workflow. The maximum working pressure is up to 6.0 bar. This outline shows a complete packing evaluation for a 14.1 L pre-packed MaxiChromAC column including HETP and Asymmetry measurements before and after ground and aircraft transportation. Furthermore, a case study for a protein purification process was performed and compared to standard stainless steel equipment.

D10 Microalgae Culture Using the DASGIP PBR4 Module for Illumination with a New Brunswick CelliGen 310 Bioreactor Stacey Willard, Eppendorf, Inc. The number of bioprocess applications for microalgae has increased in recent years, particularly in the field of biofuel production. The global biofuels market is predicted to reach US$100 Billion in 2014. As a result, microalgae bioprocess applications have the potential to expand into a multi-billion dollar sector of this growing global market. The combination of the New Brunswick CelliGen 310 benchtop autoclavable bioreactor and the DASGIP LED Illumination System creates a bioprocess setup which is capable of supporting high density microalgal growth. Using the stand-alone DASGIP PBR4 Module, LED illumination spectra and intensities can be controlled for optimal support of all types of chlorophylls and carotenoids. For this study in which high density culture of up to 1.5x107 cells/mL was achieved, the unicellular marine alga, Dunaliella tertiolecta, was used.

Recovery & Purification

E1 Efficient separation of antibody light chains from bi-specific antibody monomer using mixed-mode sorbents Catherine Allioux, Pall Life Sciences Bi-specific antibodies are currently considered as one of the most promising class of next generation therapeutic molecules and in the coming years a growing number of such products are expected on the market. Purification of bi-specific antibodies presents unique challenges compared to that of monoclonal antibodies, and chromatographic sorbents will therefore have to answer those specific requirements. In the present study, the use of mixed-mode sorbents as capture step for the purification of a bi-specific antibody was investigated, with specific focus on the separation of the abundant antibody fragments (light chains) from the monomeric form. Three mixed-mode sorbents were evaluated for their performance in antibody light chain elimination. Our data indicate that two out of the three evaluated sorbents allowed efficient removal of antibody light chains thanks to the possibility to selectively elute antibody from the column while retaining fragments on the resin. Up to 97% pure monomers were recovered after only one purification step using mixed-mode sorbents while the initial monomer purity was about 30%. Concomitantly, satisfying recovery and efficient CHOP removal (> 1log) were obtained. Altogether, these data reveal that mixed-mode chromatography is a powerful tool to address the future challenges of purification of the growing bi-specific antibody class of biomolecules.

E2 Variability of Triton X-100 impacts product solution clarity during solvent/detergent treatment David Briggs, Avid Bioservices Ensuring that high quality raw materials are used in cGMP manufacturing is critical to achieving reproducible process performance. Viral inactivation using solvent-detergent is commonly used in production processes for biologics. The visual appearance—specifically solution clarity--of a solvent-detergent treated product pool can be variable after addition of a Triton X-100/TnBP mixture. To determine the cause of this variable appearance, we focused on the role of Triton X-100 in preparing this mixture. We established that (a) the cloud point of the detergent is lowered substantially by the presence of TnBP and buffer components, and, as a result, (b) the Triton X-100/TnBP mixture specified for this viral inactivation step becomes opaque very near to ambient temperature. Variation in the quality of lots of Triton X-100 appears to cause this issue. To assure consistency in manufacturing, a high grade of Triton X-100 with traceability and extensive analytical characterization was identified and implemented. This measure has resulted in consistent solution clarity at the Triton X-100/TnPB step in cGMP manufacturing.

E3 Continuous Downstream BioProcessing by Coupling CadenceTM Single-Pass TFF with Chromatography Steps Catherine Casey, Pall Life Sciences Pall's patented SPTFF technology is enabling continuous bioprocessing by eliminating the conventional TFF recirculation loop and achieving 2-20X concentration factors in single pass. Since SPTFF can be a key process intensification tool and operated virtually anywhere within a given bioprocess, this work explored coupling SPTFF with affinity, ion-exchange and membrane chromatography steps and evaluated the versatility and robustness of its performance. The ability of the SPTFF module to stably concentrate four different feedstocks (Fab, mAb, Bovine IgG, and alpha-amylase) by a factor of 4-5X prior to the chromatography load step was demonstrated. Further testing performed with the Fab in E.Coli periplasmic extract utilizing MustangS XT Acrodisc® indicated that the process could be operated over a broader range of operational productivity within 4-11X. Coupling SPTFF with chromatography steps not only reduces chromatography load times and/or results in noticeable materials savings, but also enables downsizing of respective downstream unit operations for significant cost savings. The smaller equipment, tank sizes, and hold-up volumes also result in smaller system footprints, which are especially beneficial for manufacturing spaces where facility fit is a concern. Indeed, reduced volumes increase the likelihood of adoption and further utilization of other single-use technologies for additional cost savings.

E4 Use of Virus-Like Particles as Live Viral Surrogates during Downstream Process Development Viral Clearance Studies David Cetlin, MockV Solutions Viral clearance is a critical downstream process attribute, however, the costs and logistics associated with live virus-spiking studies make data difficult to attain during process development and characterization. Non-Infectious virus-like particles (VLPs) that mimic the physiochemical properties of natural viruses could be utilized to generate this data. VLP's of Mouse Minute Virus (MMV) were created as "viral surrogates" and used as "spiking" agent in mock viral clearance experiments. The VLPs were produced by cloning the MMV capsid gene, VP2, into a baculovirus expression vector, expressing it in Sf9 culture and purifying the resulting particles through density centrifugation. A physiochemical comparison of the VLPs' surface charge and size was made against MMV. Quantitative ELISA assays were developed utilizing natural and recombinant VLP surface epitopes. A viral-spiking study was conducted using MMV VLP as the spiking agent. Spiked NS0 supernatant was purified through a Q-SFF column. Load and flow through pool collections were analyzed for VLP quantity via ELISA. This data was compared against a standard curve generated using a dilution series of VLP and hence, LRV was determined. The results show promise that VLPs could be employed during process development and characterization as a tool to predict viral clearance efficacy.

E5 CaptureSelect™ technology: Introducing one-step selectivity in the purification of biological products Frank Detmers, Thermo Fisher Scientific Affinity chromatography is one of the most effective methods for purifying protein therapeutics. The CaptureSelect™ ligand technology addresses protein purification challenges and provides a platform approach by introducing a highly selective capture step for primary recovery purification. These solutions offer unprecedented specificity to the target protein ensuring mild elution conditions for sensitive proteins at any scale and independent from the feedstock. Author: Dr Frank Detmers, Director Ligand Application, Bioproduction, Thermo Fisher Scientific

E6 Host Cell Protein (HCP) and DNA Removal through Clarification Methods Sandra Echeverry, EMD Millipore Depth filters are primarily used to remove cells, cell debris and colloids during the clarification of the cell culture. Due to their adsorptive properties other impurities such as host cell proteins (HCP) and DNA can also be removed with these filters in various steps of the purification. Although some of these applications have been explored, a thorough exploration of the capabilities of this technology has not been reported. In this study we focused on understanding the typical performance for a given depth filter and implementing process strategies; including operation in series to maximize the HCP and DNA adsorption.

E7 Characterization of CaPure-HA, a new hydroxyapatite resin for the purification of monoclonal antibodies (mAbs) and other biomolecules William Evans, Tosoh Bioscience LLC CaPure-HA is a new, mixed-mode chromatography resin which is a macroporous, spherical form of the hexagonal crystalline structure of hydroxyapatite (HA, Ca9(PO4)6•Ca(OH)2) that has been sintered at high temperatures for increased stability. Here we present the results of the separation of proteins and a mAb using CaPure-HA resin. Column efficiency testing with both dry-pack and flow-pack columns yielded reasonable HETP and asymmetry values. The separation of three standard proteins was tested, and no significant variations in elution profiles were observed between lots. CaPure-HA has demonstrated dynamic binding capacity of greater than 20 g/L for lysozyme at residence times as low as one minute, and greater than 30 g/L for human IgG at residence times as low as 2 minutes. CaPure-HA shows a strong linear pressure flow correlation. This resin yielded high resolution separation of dimer and higher order aggregates from monoclonal antibody monomer. The resin was found to maintain dynamic binding capacity over 100 cleaning cycles with 1 N NaOH. In conclusion, this study shows that CaPure-HA is a useful, robust, alternative method for the purification of monoclonal antibodies (mAb) and other biomolecules.

E8 A Novel High Productivity Metal Affinity resin Johan Färenmark, Ge Healthcare Biosciences Immobilized metal affinity chromatography (IMAC) is a well-established technique utilizing certain amino acids, such as histidine, cysteine and tryptophan, that can form complexes with transition metal ions such as zinc, copper and nickel. Several of the IMAC resins developed in the late 1980's and early 1990's have since then established themselves as useful purification tools in large scale processes worldwide, but sometimes with limited throughput and productivity due to the pressure / flow properties of the base matrices employed in the design. Current demands for higher productivities and often also fine-tuned selectivity's have triggered the development of both higher throughput resins but also with unique and tailored selectivity. In this poster the development and industrial collaboration is presented which generated Capto Chelating. Data will be shown demonstrating the higher productivity as compared to previous generation metal affinity resins.

E9 Manufacturing Technologies and Case Studies to Increase Efficiency in Clinical or Commercial Production Chris Forespring, MedImmune Thorough evaluation of emerging technologies is a key determinant for identifying process improvement opportunities in existing and future bioprocess facilities. Successful implementation through process coupling and/or elimination of non-value added processing steps could result in both novel facility-fit solutions with alternative processing options and provide major cost savings at clinical and commercial scales. In this context, a collaborative study has been undertaken to demonstrate the use of Single-Use Inline Concentrator (ILC) linked to several potential processing steps such as perfusion, pre-capture chromatography, in-process volume reduction, and UF/DF. ILC is a disposable, self-contained, and easy to use, single-pass tangential flow filtration (TFF) device. The feasibility and performance of ILC modules were successfully evaluated and demonstrated over a wide range of feed streams at varying concentrations and process temperatures. ILC technology is an important addition to the process development tool-box for platform process evaluation, which could potentially remove constraints in existing facilities and increase the flexibility of manufacturing facilities by staying in disposables space. This presentation will cover application data with industrial molecules and the impact on the existing processes through detailed case studies.

E10 The Benefits of High Performing Chromatography Resins Including POROS® XQ, A New Strong Anion Exchanger Christine Gebski, Thermo Fisher Scientific POROS resins offer unique performance attributes and drive benefits to downstream processing. POROS XQ is the newest addition to our product portfolio. This high capacity, salt tolerant, high resolution anion exchanger enables unique selectivity, higher product yields through better separation and reduced cost of goods. Applications data will be used to demonstrate the benefits of POROS resins to capture and polish chromatography and process modeling will be used to demonstrate cost of goods improvements and process efficiencies that can be realized.

E11 Nanofibers for high productivity downstream processing Oliver Hardick, University Ciollege London • Payers worldwide are becoming ever more price sensitive and successful exploitation of the emerging and biosimilar markets will require a rethink of the current production strategies to re-focus on cost of goods, flexibility and 'developability'. • Chromatography is moving from a 'one size fits all' bead based process to one of increasing product stratification as new entrants launch technically specified products to address specific process issues.

E12 The Renaissance of Expanded Bed Adsorption? Martin Hoffman, Biotechflow 20 years after development, Expanded Bed Adsorption chromatography fell out of favour in Bioprocessing. Proposed improvements in process efficiency- obtaining high yields from unclarified feed stocks- were compromised by column design and process run failures; in part caused by blocked meshes, lipid breakthrough and demand for high buffer volumes producing high eluting volume. Current media optimize Dynamic Binding Capacity within the expanded bed. Coupled with improved inlet design, increased bed densities are achieved, which permit high flow rates (450 cm/hr) and throughputs. Direct product capture reduces unit costs by 66%, and labour cost and process time. Comparisons with packed bed chromatography result in higher product yield and 43% reduction in buffer volume. Fully-scalable, cGMP EBA column design improvements prevent vortex formation, whilst ultrasound technology enables contactless monitoring of process dynamics and flow rate via feedback to the skid; both factors maximize expanded bed stability. Reduced column footprint and height requirements increase versatility. In recent years, step changes in incubation techniques, media design, column technology and sensor applications have enabled proven improvements in EBA process efficiency and operations. The initial promise may now be considered as surpassed, and a renaissance of Expanded Bed Adsorption Chromatography should be proposed. (200)

E13 Early phase PD concept for downstream Hidenori Inaba, Astellas Pharma Inc Astellas is working on process development (PD) scheme based on QbD approach. This poster presentation will introduce Early stage PD concept for MAb purification. We implemented risk assessment and setting of CEC elution conditions. The method is scaled up for manufacturing scale excellently.

E14 Natrix Hydrogel Technology: A Fully Disposable Toolset For Biomolecule Purification Renaud Jacquemart, Natrix Separations The rising demand for biopharmaceutical products has driven the need for greater operational speed and efficiency for biomanufacturing. Existing downstream process technologies have struggled to achieve these metrics, but recently new solutions have become commercially available. Natrix Separations is working to provide a fully disposable, single-use (per batch) toolset for biomolecule purification that offers superior throughput and flexibility with no compromise on quality. The currently available tools employ well-known ion exchange binding modalities, but others (including affinity), are under development. Natrix membranes all feature a high density of highly accessible binding sites that enable high productivity capture, intermediate purification and/or polishing operations for monoclonal antibody, recombinant proteins and vaccines GMP manufacturing. The results presented here demonstrate the performance of Natrix HD membranes for mAb purification.

E15 Design of a novel agarose based resin platform Hans J, Purolite The first agarose based chromatography beads were introduced by Hjerten already in 1962. Fifty years later beaded agarose has become the dominant resin for protein purification and is extensively used, from research scale in sub ml volumes, to full scale manufacturing in > 500 litre chromatography columns. However, since the introduction of Sepharose 6 Fast Flow in 1982, relatively few homogenous agarose based matrices designed for large scale manufacturing, have entered the market. The focus of recent research has been more on increasing capacity and selectivity through different grafting technologies and ligand developments rather than improving the inherent properties of the agarose beads. In this paper we have used new technologies to produce beaded agarose of different particle sizes ranging from 40 – 100 µm. The objective has been to improve pressure flow properties while creating a porosity structure optimal for protein chromatography. The result indicate that there are good possibilities to design homogeneous agarose resins for large scale manufacturing, with significantly improved pressure/flow properties, capacity, and resolution compared to what is commercially available today. Application and characterization data from a set of novel agarose based ion exchangers will be presented.

E16 Development of a technology platform for purification of antibodies – Case study with a biosimilar model molecule Benjamin Kester, Cook Pharmica LLC In recent years, platform purification processes have been applied to monoclonal antibody and antibody intermediate biologic development. The purification steps in a standard three-column process consist of Protein A affinity purification, followed by ion exchange, and a polishing step. The work presented here focuses on development of a rapidly optimized downstream process using a technology platform. Protein A wash steps were optimized to reduce host-cell proteins and other process related impurities. Protein A intermediate was further processed to develop a cation exchange step. Design of experiments was utilized to rapidly arrive at favorable conditions for use in a three-column or two-column process. Follow-up experiments were carried out confirming the selection of critical processing parameters. Finally, TFF was optimized using AKTA Crossflow. Optimization experiments were carried out at initial concentration and final concentration to define operating flow rate and transmembrane pressure. Additionally, a diafiltration time optimization analysis was performed to define the optimal concentration for diafiltration. The process was confirmed at two different scales and product quality was shown to meet specifications. Unicorn automation and analysis software included with the AKTA Crossflow expedited the process and facilitated process understanding.

E17 Efficient Purification of PCB-101 by Crystallization in few simple steps Saraswathi Mandapati, ProCrysta Biologix Inc Protein purification methods have improved to a greater extent and reached a plateau with widely accepted affinity tags, kits and protocols for smaller quantities. However, booming biologics development with higher cell densities demanding for high throughput protein purification techniques, where often limited by column chromatography. Efficient and systematic approaches developed for bulk crystallization of different classes of biopharmaceuticals including monoclonal antibodies can serve as a solution to overcome these limitations in protein purification. Protein crystallization is applied to purify many different classes of therapeutic macromolecules. Through this innovative approach biomolecules can be purified directly from crude sources such as cell lysate, culture media or clarified milk with higher purity and for better recovery compared to conventional chromatography techniques. This process is highly reproducible, scalable and also reduces bacterial endotoxins and viral load. PCB-101 an enzyme, for treating tuberculosis was expressed in Escherichia coli with and without His tag. Purification through chromatographic method resulted in poor recovery and purity, whereas His tag decreased solubility and activity. A fully active protein without His tag was purified by crystallization technique in three simple steps with >95% purity. Similarly, other biomolecules such as enzymes, monoclonal antibodies, polyclonal antibodies, hormones and fusion proteins were successfully crystallized from crude sources. Protein crystallization, a single technique, can become an economical tool for purification and stable formulation of bio-therapeutics.

E18 Improved Protein G for the Purification of Antibody Fragments Dai Marata, Kaneka Corporation Streptococcal Protein G is well known as an affinity ligand for all subclasses of human IgG. It binds strongly to the Fc region and weakly to the Fab region. Low Fab recovery, attributed to weak binding, has meant that Protein G media has not been widely adopted in the industrial chromatography arena for Fab purification. However, Protein G has the potential to be a widely used affinity ligand for Fab purification if the binding can be improved. In this study, we present data for our improved Protein G which was engineered using our ribosome display system, PUREfrexRD (GeneFrontier Corporation) for in vitro screening and had strong binding to Fab. The association constant (KA) of engineered Protein G increased from 30 to 70-fold compared to wild type Protein G. The improved Protein G with highest KA was immobilized to agarose media and its performance was evaluated. Our engineered Protein G media gave drastically improved Fab recovery, more than 90 %, much better than recoveries of less than 10% for commercially available Protein G media. Our results demonstrate that engineered Protein G is useful as an affinity ligand and can be applied to your purification process development platforms for Fab's.

E19 Improving Safety and Efficiency in a Demanding Pilot Plant Environment Ron Massicotte, Genentech / Roche Illustrate & explain several improvements that have enhanced safety and efficiency in a busy pilot plant environment.

E20 Applying QbD Based Approach to the Characterization of a Downstream Process - A Case Study Yi Jian Miao, Shire A systematic Quality by Design (QbD) approach was applied to the characterization of the downstream process for the manufacturing of a therapeutic protein product. The product critical quality attributes (CQAs) were first determined. For every downstream unit operation, a FMECA process parameter risk assessment against process performance and product quality was performed. Following risk evaluation and prioritization, the higher risk parameters were included in a multivariate high resolution DOE study to evaluate the parameter effect and potential interactions. Univariate studies were also performed for selected process parameters. The studies linked process parameters to CQAs and process performance, and generated unit operation heat map for the CQAs evaluated. Appropriate control strategy can then be developed to ensure manufacturing success.

E21 COMPARISON OF BINDING CAPACITY OF TOYOPEARL AF-rProtein A HC-650F AFFINITY RESIN AT VARYING BED HEIGHTS AND CONSTANT COLUMN VOLUME Kelly Motter, Tosoh Bioscience Protein A affinity chromatography most often serves as the initial capture step and is typically the most expensive step in the downstream production of mAb bio therapeutics. TOYOPEARL AF-rProtein A HC-650F affinity resin utilizes a recombinant, alkaline stable proteinA ligand with increased dynamic binding capacity and excellent pressure-flow characteristics. A variety of column dimensions in terms of internal diameter and length are used by scientists when scaling up protein purification procedures to optimize speed, yield, and recovery to make production as efficient and cost effective as possible. Here, we show that altering column dimensions to favor a shorter, wider column will maintain equal binding capacities when compared with a longer, narrower column of the same total volume. This will allow for both ease of scale up and improved flow characteristics. All columns tested achieved recovery of 96-99% of loaded sample. Additional data demonstrates the resin's capabilities of removing both host cell proteins and DNA.

E22 Evaluation of different commercially available Protein A resin on their DBC to Nanobodies Masayoshi Nagaya, JSR Life Sciences Nanobodies are a novel class of antibody-derived therapeutic proteins based on single-domain antibody fragments for a range of serious human diseases including inflammation, hematology, oncology and pulmonary disease. Different purification strategies are applied by the industry for the purification of these molecules. Although Nanobodies lack the Fc Part of conventional antibodies, some Nanobodies do bind to Protein A. The nature of this binding is not fully understood today. In this work several commercially available Protein A resins have been tested on their ability to purify Nanobodies. The tested Protein A resins all have different engineered Protein A ligands which may influence their binding interaction to Nanobodies. The highest binding capacity was obtained with Amsphere Protein A JWT203, a novel protein A resin developed by JSR Life Sciences (Japan).

E23 Improved design of Continuous Countercurrent Tangential Chromatography (CCTC) for Single-Use mAb Capture Boris Napadensky, Chromatan Chromatan's CCTC is a column-free mAb capture technology that provides a single-use continuous unit operation that overcomes many limitations of packed bed chromatography. CCTC replaces the stationary phase of a packed column with a moving slurry that is continuously pumped through several cascades of static mixers and hollow fiber membranes. This work showcases a new system design which resulted in significant simplification and scale down from the previous versions of CCTC. In collaboration with Regeneron, mAb capture and purification from a clarified CHO harvest was performed with run time of 8 hours and batch volume of 2 Liters. The system showed >90% yield while operating at low system pressures (< 10 psi). 3X improvement in HCP clearance, and 30% improvement in aggregate clearance was shown when compared with the packed protein A column

E24 Direct Quantification of Residual Host Cell DNA Nancy Ngo, Bio-Rad Laboratories Many therapeutic proteins and vaccines are manufactured using bacterial and mammalian host cells. Manufacturing processes are prone to leaving biological impurities from these cells, such as host cell DNA (HCD). The presence of HCD in drug substances poses safety concerns and must be removed to ensure product quality and safety. Regulatory agencies, such as the U.S. Food and Drug Administration (FDA) and the World Health Organization (WHO), have provided guidelines defining acceptable levels of HCD allowed in final drug products; the upper limit is 100 pg/dose and 10 ng/dose as stated by the FDA and WHO, respectively. Therefore, the method to quantify residual HCD and monitor DNA clearance should be highly sensitive to meet these regulatory requirements. Here, we introduce a highly precise and sensitive method for residual HCD quantification, without the need for DNA extraction, using droplet digital PCR (ddPCR). We created a panel of test matrices to simulate various process intermediates and analyzed these samples using ddPCR. Spiked-in DNA can be fully recovered even in complex matrices with high precision and femtogram-level sensitivity. The results from our study clearly show the effectiveness of ddPCR in detecting residual HCD by a direct method and without the need for DNA extraction.

E25 Characteristics of small particle polymer media for cation exchange chromatography Shinya Nozaki, Mitsubishi Chemical Corporation Cation exchange chromatography is a well-established unit operation in the downstream processing of monoclonal antibodies. In this work, our cation exchange media, ChromSpeed S series, are introduced and their adsorption and separation characteristics are discussed. The base matrix is highly porous rigid polymethacrylate, and the average particle diameter is 30 micrometer for S101, and 60 micrometer for S103, respectively. High dynamic binding capacities and good separation were observed when they were applied for human antibodies, presumably owing to rapid intra-particle diffusion rate. As purification media, ChromSpeed will meet the demands of biopharmaceutical manufactures by improving productivity and reducing process time.

E26 High Capacity Depth Filtration Kristina Pleitt, Gallus Biopharmaceuticals The clarification process — in which cellular debris are separated from the product stream — is a critical and often overlooked step in biomanufacturing. Depth filtration has been widely used for the removal of cell debris as well as other soluble and insoluble contaminates. Recent advances in cell culture technology have resulted in consistently higher cell density feed streams (> 25 x 106 cells/mL) which pose a great challenge for biomass and soluble species removal using traditional depth filtration. To allow for depth filtration to be used as a harvest strategy for cell cultures having cell density, a combination or pre-treatment and next generation depth filtration was investigated. Pre-treatment of unclarified cell culture harvest with acid or flocculent has been shown to reduce the impurity load prior to downstream purification via the precipitation of contaminating impurities. Commercially available flocculent pDAMAC has been used to reduce the levels of negatively charged impurities in untreated feed streams. In this study, cell culture harvest was pre-treated with acid or polyionic flocculent (pDADMAC) prior to filtration with Clarisolve depth filters from Millipore. This filtration strategy was compared to the traditional depth filtration using the Millistak+ Pod depth filters from Millipore. The performance of the two strategies with respect to filter loading, filtrate turbidity and yield will be presented. In addition, an analysis on the impact of this technology on scale-up and economics will be discussed.

E27 Chromabolt® Prepacked and Pre-Validated Columns: Three-Resin Validation Approach Santosh Rahane, EMD Millipore Pre-packed chromatography columns have demonstrated their value in ease-of use to the process development scientists, especially in pre-clinical and early clinical phase. Chromabolt® products are a family of pre-validated columns prepacked with EMD Millipore chromatography resins, designed for early clinical stage manufacturing. The Chromabolt® columns are available in 3 sizes – 10, 20 and 32 cm inner diameter (i.d.), and free up end users valuable time and resources by eliminating manual packing and cleaning. The column manufacturing process, which involves automated packing and sanitization in clean room produce ready-to-use columns with highly consistent (more than 99% of success rate) HETP, Asymmetry and flow properties. The columns' ergonomic design enables both the safety and ease of use. Additionally, carefully designed shipping method ensures that the columns arrive at end users' facilities safely and intact as proved by our shipping validation tests. The current work with Chromabolt® columns focuses on the recently launched columns that offer prepacked columns of Fractogel® COO, Fractogel® SE Hicap and Eshmuno® Q resins in addition to the current Chromabolt® columns offering prepacked Fractogel® TMAE Hicap, Fractogel® SO3 and Eshmuno® S resins. The development of the Chromabolt® columns with the new resins was achieved by adapting a three-resin approach in combination with the Taguchi model for statistical experimental design. This approach allowed us to conduct a complete validation of all the three resins at several packing conditions and in all i. d. columns. The success of standard shipping validation tests ensured column integrity after shipping. In summary, the three new resins were successfully accommodated into the Chromabolt® formats without any change in the current packing processes and with high consistency of packing method and characteristics.

E28 Polishing of Monoclonal Antibodies using a Polymer Grafted Cation Exchanger Jonathan Royce, GE Healthcare Life Sciences The polishing steps of monoclonal antibodies present several challenges. One of the major goals of the purification process is to reduce the amount of aggregates. Typically, cation exchange chromatography is included in the purification scheme to address this problem. A new grafting technology has been developed, enabling attachment of polymeric surface extenders with cation exchange functionality onto agarose beads for use in polishing of monoclonal antibodies. The performance of these polymer grafted cation exchangers has been investigated and will be presented. The polishing step for different monoclonal antibodies was optimized regarding buffer composition and pH using 96-well filter plates and later confirmed in packed bed using column format. Yield, aggregate- and HCP-removal was monitored throughout the study. Screening conditions for binding, selectivity and optimization of elution conditions are presented, as well as comparison with CIEX chromatography media on the market.

E29 Maximizing Viral Clearance for High Titer Validations Alexander Schwartz, Asahi Kasei Bioprocess Virus filtration is considered to be one of the most robust viral clearance processes. Contemporary small virus filters reliably achieve greater than 4 logs of reduction of non-enveloped viruses in most processes, allowing greater focus on development activities related to maximizing volumetric throughput and yield. Nonetheless, the recent development of high titer spiking stocks has led to increased challenges for ensuring high virus removal while maintaining high product throughput. In order to further investigate the effect of high titer spiking stocks on viral clearance with Planova™ filters, we performed clearance studies using PP7, a small non-enveloped bacteriophage, as a model for viruses such as parvoviruses. Logarithmic reduction value (LRV) was found to be dependent on the overall challenge titer, and residual virus in the filtrate increased significantly when the viral retention capacity of the filter was exceeded. Currently, our recommendation is to perform viral clearance studies using a total virus challenge of no more than 11 log PFU/m2 to mitigate the risk of over-spiking the filter. However, for those processes with Planova filters for which higher LRV is critical, we propose using two Planova filters in series. Using this filters-in-series strategy, we were able to achieve over 5-fold increase of product throughput with complete PP7 clearance (LRV ≥ 7 ). This alternate process, with an optimized control strategy, takes full advantage of the reliability and performance of Planova filters while achieving high viral clearance that is only possible with high titer virus spiking.

E30 An Optimized Approach for Anion Exchange (AEX) Chromatography to Enhance Productivity and Decrease Cost of Goods Yuyi Shen, XOMA(US)LLC Yuyi Shen*, Jimmie Pagsolingan, Jeffrey Bettencourt, Shelly Parra *Corresponding author Abstract: Anion-exchange (AEX) products (resin/ membrane adsorbers) are commonly used as a monoclonal antibody purification polishing step in process flow-through (FT) mode for reduction/removal of process impurities such as DNA, host cell proteins, endotoxins . Using a high-performance AEX resin at high operating flow rates and shorter bed heights, can significantly increase throughput while providing robust impurity clearance and high yields. Combining this approach with the convenience of pre-packed column will provide a more productive unit operation within the manufacturing process, reducing cost and time. The poster will show how combination of AEX resin and utilization of pre-packed and pre-qualified column can drive significant reductions in process time and costs compared to the traditional approach of in-house packing and qualifying the column for purification of monoclonal antibodies.

E31 Bench top multicolumn continuous chromatography: an enabling platform for highly productive mAb purification Alla Zilberman, Semba Biosciences Simulated moving bed (SMB) chromatography and its variant multicolumn continuous chromatography (MCC) have the potential to elevate the industrial chromatographic platform by conversion of conventional batch processes to more efficient continuous processes. The high productivity, recovery and purity achieved by SMB chromatography on an industrial scale for small molecules hold promise for biomolecule manufacture. We used a lab-scale SMB device to perform continuous Protein A Capture (PAC) for the purification of mAbs. Rather than using one large adsorbent column and a sequential batch protocol, the SMB device uses eight small columns, multiple input and output streams, and a continuous protocol in which several protocol steps occur simultaneously in different columns, analogous to the "row, row, row your boat" song sung in rounds. Flexibility in programming column and flow configurations enable optimization of purity, yield, and adsorbent utilization. Unlike batch, SMB-PAC is primarily a volume-driven process in which a given volume of feed material can be processed with the same amount of adsorbent, irrespective of titer. As mAb titers increase, flow rates and productivity increase. To take full advantage of the potential productivity gains afforded by SMB-PAC for processing high titer mAb, per column residence times must be decreased while maintaining efficient capture. For this study we purified a humanized IgG1a mAb from CHO culture fluid using the Octave System in a SMB-PAC process and an AKTA System in a standard batch PAC process. We compared the performance of an agarose-based Protein A adsorbent with two polymethacrylate-based Protein A adsorbents in both processes.

* This list of posters may not be the final displayed on-site at the event. Changes/additions may occur prior to the event.

Vaccine Development & Production Summit

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