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THE Industry Meeting Place to Exchange Real-World Solutions to Improve Speed, Cost and Quality
Conference: October 20-23, 2014 · Exhibition: October 17-19, 2014 ·
2013 Poster Presentations
2013 Poster Presentations
Dedicated Poster Viewing Hours
Poster presentations will be on display at all times during exhibit hall viewing hours. Poster presenters may stand by their posters at any time, but to ensure that attendees are able to meet poster presenters at specific times during the conference, we have designated the following days/times as "Dedicated Poster Viewing" Hours.
For Even Numbered Posters
Tuesday, September 17, 2013
Wednesday, September 18, 2013
For Odd Numbered Posters
Wednesday, September 18, 2013
Thursday, September 19, 2013
General Exhibit Viewing Hours
Tuesday, September 17, 2013
Wednesday, September 18, 2013
Thursday, September 19, 2013
Learn new approaches to your research programs
Updated: September 6, 2013
Analytical and Quality
A1 Introducing a New BLI Instrument Focused on Throughput and Flexibility : The Octet HTX System Dominic Andrada, ForteBio - A Division of Pall Life Sciences Biolayer Interferometry technology has become well established over the past decade for protein quantitation and kinetic characterization of molecular interactions. The non-fluidics based Dip and Read™ format of the Octet instrument line lends itself to highly multiplexed reading, and thus to higher throughput than other label-free techniques. As with the entire Octet line of instruments, the Octet HTX system is compatible with the full range of over 18 biosensor types including protein A, streptavidin, anti-His6-tagged and anti-species coated biosensors. In this poster, we present key applications using the newly developed Octet HTX instrument
A2 Enhanced 2D Electrophoresis and Western Blotting Workflow for Reliable Evaluations of Anti-HCP Antibodies Sara Heitkamp, Bio-Rad Laboratories Biologics and biosimilars can contain traces of proteins from the host cell line, which need to be minimized to prevent adverse immune reactions. The presence of host-cell proteins (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCP's in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting. Tools that simplify, standardize and accelerate 2-D electrophoresis and blotting have become available. Advanced electrophoresis and blotting instrumentation, stable and reproducible pre-cast gels, next-generation imaging and image analysis software all allow this assay to be performed in a reproducible manner in a relatively short time. We have undertaken to demonstrate this using a commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2 D electrophoresis procedure resulted in highly reproducible spot patterns and entire procedure was completed in less than two days. The software used (PDQuest) enabled the straightforward generation of percent coverage values for the antibody when used to probe both CHO cell lysate and CHO cell secreted protein.
A3 The Importance of Thresholding in Imaging Analysis of Protein Aggregates Lew Brown, Fluid Imaging Technologies, Inc. Dynamic imaging particle analysis (DIPA) shows much greater sensitivity to transparent particles, such as protein aggregates, than light obscuration can. While not yet fully accepted in industry compendia, DIPA is being used increasingly in the formulation process for characterization of sub-visible particulates in biologics. DIPA measures particle size and shape by first creating a binary image based upon a defined threshold from the background value for each pixel in the image. The resulting binary image is used for all particle measurements, so the choice of thresholding technique is critical to the validity of the measurements. This poster will demonstrate how different thresholding techniques can produce dramatically different characterization of protein aggregates. To do this, a base set of camera images are acquired for a therapeutic protein containing aggregates. These "raw images" can then be run through image processing software using different thresholding techniques to quantify and measure the aggregates contained. By using the same set of images for each thresholding process, we insure that any variation in results can only be caused by the thresholding process. The results will show striking differences caused by the thresholding variation, thereby supporting the importance of proper thresholding when using DIPA
A4 Using Helium Integrity Tester to Increase Assurance of Sterility of Single-use Assemblies Carla Conant, ATMI LifeSciences The rapid adoption of single-use bioprocessing technologies continues to raise concerns about integrity of flexible containers and assemblies. The poster discusses inadequacies of the existing integrity testing methods for flexible containers and presents a novel method, of using helium trace gas for detecting defects down to 10 µm in prior-to -use-mode. A microbial aerosol challenge study conducted on flexible sterile containers revealed that defects as small as 12 µm are capable of causing sterility breach while defects smaller than 10 µm did not show microbial ingress under the test conditions. Data will be presented demonstrating detection of 10 µm defects for single use bags, complex manifolds and assemblies. Relationship between the probability of sterility breach and defect size will be discussed.
A5 Evaluation of the Cedex Bio and HT Metabolite Analyzers for Cell Culture Process Monitoring Caroline DiCesare, Genzyme, a Sanofi Company Caroline DiCesare†, Tim Johnson†, Adam Borey‡ †Commercial Cell Culture Development, Genzyme Corporation, a Sanofi Company, 45 New York Avenue, Framingham, MA 01701 ‡Cell Culture Operations, Genzyme Corporation, a Sanofi Company, 68 New York Avenue, Framingham, MA 01701 Abstract Metabolic intermediates and by-products such as glucose, lactate, glutamine, glutamate, and ammonium are critical cell culture process indicators. The concentrations of these metabolites are often monitored to assess culture performance. This information is frequently used to make decisions during the development and operation of cell culture processes. As such, a metabolite analyzer that is accurate, precise, and reliable is a critical tool in gathering this information. Currently, there are numerous vendors and detection technologies available in the marketplace, including amperometric and photometric based technologies. This poster will present the evaluation results of three commercially available metabolite analyzers: Cedex Bio, Cedex HT and YSI for their accuracy, precision, robustness, ease of use and maintenance. The Cedex platform utilizes a photometric technology whereas the YSI platform is based on amperometric measurements. Our results showed that the Cedex Bio and Cedex HT performed comparably to the YSI 2700 in terms of accuracy and precision for glucose, lactate, glutamine, and glutamate. We will also discuss other pros and cons in terms of additional applications, robustness, ease of use and maintenance of these three analyzers.
A6 Accurate mass structural elucidation of biomarkers in yeastolate by UPLC/MS (qTof) and composition profiling by multivariate analysis to reduce variability in cell culture media Barry Drew, SAFC Yeastolates are complex and undefined supplements containing a mixture of proteins, peptides, amino acids, and other neutraceuticals. Yeastolates are commonly used because they are not an animal derived raw material and have been shown to increase growth and/or productivity of bacterial, insect, and mammalian cell cultures. However variability in biomass and growth production as much as 50% has been observed. To reduce this variability, analytical methods such as UPLC-MS can be used to discern potential biomarkers in yeastolate lots to screen potentially poor performing lots from being used in the manufacture of cell culture media. Yeastolate lots from several vendors were analyzed by UPLC-MS (qToF). Multivariate analysis of the accurate mass compositional profiles were performed and associated with specific growth characteristics from an EB66 cell line. Good and poor performing lots within a vendor were compared via discriminant analysis (OPLS-DA) and biomarkers identified as contributing to the compositional difference were identified by elucidating the structure via MSMS analysis.
A7 Optimization of O-glycans Stuctural Analysis by Nonreductive Alkaline Beta-elilmination Guillermina Forno, Zelltek S.A. Although O-glycosylation is an important post-translational modification, the O-glycans release and the subsequent analysis remains a challenge. Nonreductive alkaline beta-elimination is a chemical method that allows N- and O-glycans release. The aim of this work was to optimize experimental conditions for selective removal of O-glycans with maximal reduction of the undesirable peeling. The studied variables were temperature, time of hydrolysis and type of reagent used to obtain the required alkalinity conditions (pH 9,50-10,0). According to the observed results, it was possible to optimize the O-glycans removal conditions with peeling minimizing using ammonium carbonate as an alkaline agent during 4 hours and keeping the temperature at 60 °C. After hydrolysis, samples were neutralized and labeled with a fluorophore. Derivatized O-glycans were analyzed by HILIC coupled to fluorescence detector. Final working conditions were established by fetuin analysis, verifying the identified structures with the bibliography. Also two well-known therapeutic recombinant glycoproteins were analyzed: human erythropoietin (rhEPO) and Etanercept (fusion protein used in rheumatoid arthritis treatment). In the case of rhEPO the structures found were Gal(beta)1-3GalNAc forms (11.6%) decorated with one (39.9%) or two (48.6%) neuraminic acid residues. The same structures were found for Etanercept, but the bisialylated form was the more abundant (71.6%). As the O-glycans structures of Etanercept have not been described in the bibliography yet, our results are a valuable contribution to this protein characterization.
A8 To Err is Human, a Case-Study on Improving Discrepancy Analysis and Right First Time Melani Gantes, Genentech Inc Human error-related deviations impact pharmaceutical companies financially through expensive write-offs for product rejects and through delays and rework. They also can impact a company's right to operate if their response to these events is inadequate. Even so, "human error" is still a leading cause when deviations are closed. At the Genentech South San Francisco manufacturing facility, it was identified that many human-factor events have corrective actions of counseling or re-training and the business processes in place did not allow for sustainable analyses of human-factor deviations. This poster focuses on the improvements made to identify true root causes for human-factor events and in turn drive robust corrective actions.
A9 Influence of HCP antigen preparation on assay sensitivity and specificity Yvonne Haberkorn, BioGenes GmbH BioGenes investigated the influence of different HCP preparations on the sensitivity and specificity of their corresponding polyclonal anti-HCP antibodies. All HCP preparations were derived from cell culture supernatant of MOCK fermented CHO-cell lines. Reactivity of obtained antibody sets was investigated by gel electrophoresis and Western blot as well as by testing of different real samples and HCP standards by ELISA. Based on these results, BioGenes developed new tools for HCP detection with improved sensitivity and specificity
A10 Differentiation of Protein Particles and Silicone Oil Droplets by Flow-imaging Microscopy (MFI and FlowCAM) and Resonant Mass Measurement (Archimedes) Andrea Hawe, Coriolis Pharma Research GmbH We comparatively evaluated flow-imaging microscopy and the recently introduced technique of resonant mass measurement (Archimedes, RMM) as orthogonal methods for the quantitative differentiation of silicone oil droplets and protein particles in the submicron and micron size range. Representative samples of artificially generated silicone oil droplets and protein particles were prepared and analyzed both separately and in defined mixtures by the different instruments (MFI4100 and MFI5200, Protein Simple; FlowCAM VS1 and FlowCAM PV, Fluid Imaging). An accurate differentiation of silicone oil droplets and protein particles could be achieved by all flow-imaging microscopy systems in the micron size-range. Clearly lower standard deviations (higher precision), were obtained by MFI5200 and FlowCAM PV due to the higher sampling efficiency, as compared to MFI4100 and FlowCAM VS1. The customized filter (MFI4100) was more reproducible and enabled reliable discrimination for more extreme mixing ratios. RMM showed highly accurate discrimination form about 0.5 to 2 µm independent of the ratio between silicone oil droplets and protein particles, provided that sufficient particles were counted. We recommend combining flow imaging microscopy and resonant mass measurement for a comprehensive analysis of biotherapeutics potentially containing silicone oil droplets and protein particles in the submicron and micron size range.
A11 Evaluation of a microfluidic procedure for determination of residual protein A at acid conditions Mats Inganäs, Gyros AB Protein A ligands may leach from chromatographic resins during purification of recombinant IgG. Quantification of residual protein A in presence of huge amounts of IgG pose significant problems due to complex formation and reduced antigen accessibility at neutral pH. Using a new microfluidic CD containing microstructures with a mixing chamber pretreatment of samples can be performed completely automatically in Gyrolab xP workstation prior to analysis. The pretreatment is performed in two steps; first the sample is acidified at pH 2.5 in order to effectively dissociate preformed complexes between IgG and protein A, secondly, the pH of the first acid mixture is adjusted to pH 2.8 at which a selected capture reagent still is sufficiently active to capture free protein A in the acidified sample. Finally the analysis is completed by adding a fluorophore labeled detecting antibody at neutral conditions. The new concept has been evaluated using using native protein A and MabSelect SuRe, respectively, and different IgG preparations to demonstrate the utility of the procedure. The procedure takes approximately 70-80 min for 48 datapoints. Samples can be analysed at 5 mg/ml of IgG in a working range of 0.37- 90 ng/ml indicating protein A can be determined at sub-ppm concentrations.
A12 Near-universal Similarity Bounds for Bioassays David Lansky, Precision Bioassay, Inc Several versions of composite and parameter-specific measures of non-similarity will be described and compared. Two approaches to sensitivity analyses yield new ways to examine the potential impact of various amounts of non-similarity and offer a good rationale for universal similarity equivalence acceptance limits. Factorial combinations of various amounts of parametric non-similarity are used in simulations to compare different candidate measures of non-similarity. Comparisons include the properties of each measure of non-similarity (i.e.; bias) and the performance of potency (i.e.; bias). A recommended modification of parameter-specific equivalence bounds for non-similarity yields nearly universally applicable good control of important non-similarity.
A13 Development of a multi-angle light scattering detection method for use during monoclonal antibody downstream purification to monitor and control the final level of aggregation Bruce Mason, Amgen, Inc. Multi-angle light scattering (MALS) has the potential to be a useful technique for the purpose of monitoring the overall amount of high molecular weight species (HMWS) present in antibody therapeutics in real time during downstream purification steps. Current practice for the purification steps when processing monoclonal antibodies is to establish a correlation between the UV absorbance at 300 nm to the levels of HMWS and then base the exclusion of HMWS on a predetermined percentage of the UV absorbance peak maximum. This method for controlling the levels of HMWS in the product has the disadvantage of not being able to directly monitor the eluted levels of HMWS. It instead relies upon the consistency of the process and requires the relative amounts of HMWS be evaluated by an orthogonal offline analytical method such as size exclusion - high performance liquid chromatography (SE-HPLC). Alternatively, MALS as a process analytical technology (PAT) would allow for real-time monitoring of HMWS as these forms elute during processing chromatography steps alleviating the need of offline SE-HPLC analysis. Here MALS was coupled to the cation exchange (CEX) purification step for an IgG2 monoclonal antibody. Proof of concept was demonstrated on an analytical scale using an AKTA Explorer system. In addition, a total of 10 pilot plant runs incorporating an analytical scale MALS detector attached to the slipstream of the pilot plant scale CEX purification were conducted. A dimensionless molecular weight ratio (DMWR) was determined and demonstrated excellent correlation with levels of HMWS.
A14 Comparison of N-Glycans in Innovator and Biosimilar Erythropoietin Preparations Using the GlycanMap® Xpress™ Assay Service Yoshiaki Miura, Ezose Sciences Inc Detailed and high-throughput characterization of the glycans present on recombinant glycoproteins remains an important challenge to researchers involved in the development and production of both innovator and biosimilar molecules. Several innovator and biosimilar versions of Erythropoietin (EPO) are available, making this a useful model for comparative studies. EPO is a complex glycoprotein with 3 N-linked sites and its clearance rate is heavily influenced by glycosylation. Five preparations of EPO, including three innovator drugs and two biosimilars were analyzed using the rapid, GlycanMap® Xpress™ assay. This assay service relies on enzymatic release of glycans from the proteins, followed by on-bead capture, washing, and derivatization to stabilize sialic acid residues. Purified glycans are simultaneously labeled and released, and then quantitated using MALDI-TOF mass spectrometry. Internal standards are added to enable quantitation. The key assay steps are integrated into a custom, 96-well robotic assay system. Proprietary bioinformatics are then used to identify glycan composition/structure and determine quantities of each glycan. Comparison of innovator molecules Eprex®, NeoRecormon®, and Biopoin® and Eprex® biosimilars Silapo® and Binocrit® yielded glycan profiles that were generally qualitatively similar but quantitatively different. Compared to the innovator biologic, Silapo had lower levels of O-acetylation whereas Binocrit had higher levels of phoshorylated glycans.
A15 Improve Manufacturing Reliability by Implementing Predictive Maintenance Kristin Scharf, New England Controls Process Industries other than BioProcess, such as Power, Pulp & Paper, and Refining, have been focused on improving equipment reliability for many years. One of the major proven comprehensive predictive diagnostic tools available is vibration monitoring of rotating equipment. As available capacity tightens and financial pressures mount in the BioProcess industry, the impact of unexpected equipment failures has become more of an issue. Clean room air handlers, refrigeration units, compressors, and large clean utilities pumps can have a significant impact on process availability if there is an unexpected failure. With vibration monitoring and appropriate analysis, equipment problems can be detected early and relatively inexpensive preventive measures can be taken to extend equipment life and prevent unexpected failures. Early detection allows repair prior to a catastrophic event. It is not uncommon to find once a program is started, that 10% of the assets can immediately benefit from monitoring and early remediation. At an average cost of $10,000/deviation, resolving deviations associated with equipment failures adds up quickly. Opportunity costs due to lost productivity and possible product loss have an even greater impact. The case study to be presented will address the types of equipment monitored, the frequency of detection of compromised equipment, the cost avoidance achieved through avoidance of unplanned down time, and the maintenance cost savings from catching issues early. Co-Authored by Frank Corden, Director of Life Sciences Industry at New England Controls
A16 High Throughput Viscosity Measurement for Assessing Biologics Formulations David Yamane, Freeslate Biologic formulations are trending toward higher protein concentrations. High concentration formulations can reach viscosities that are not viable for commercialization. Viscosity is consequently becoming a critical consideration during the assessment of potential formulations. Freeslate has developed the CM Protégé Biologic Formulation Assessment System which has a High Throughput Viscosity Measurement Module that enables viscosity measurement of samples using a volume of 100 microlitre and has a throughput of three minutes per measurement. The system can be used to measure both Newtonian and non-Newtonian fluids. The system has been optimized for the measurement of viscous samples (1 cP to 100 cP) across a temperature range from 4 °C to 40 °C. To evaluate the performance of the High Throughput Viscosity Measurement Module on the CM Protégé Bioformulation Assessment System, Bovine Serum Albumin, multiple concentrations of a monoclonal antibody solution measured at multiple temperatures and 24 different monoclonal antibody (mAb) formulations, were tested. Measurements were fast, ~3 minutes/sample, consumed only 100 microlitre of sample and showed excellent reproducibility.
A17 Protein Aggregation, Sub-Micron, and Sub-Visible Particle Population - Data Management and Analysis Clark Merchant, Lumetics There are currently no fewer than six entirely unique sub-micron/sub-visible particle analysis technologies regularly applied to characterize protein formulations (DLS, NTA, RMM, LO, Flow Microscopy, and Electro-zone Sensing). Each has its own operating principal, vendor-specific implementation and range of instrument models. Due to differing measurement principals, the technologies often report contradictory results and present these results in different formats. Aggregating, processing, and interpreting large volumes of orthogonal data to meet regulatory requirements and quality-by-design initiatives can therefore be quite challenging.
A18 Evaluation of a microfluidic procedure for determination of residual protein A ligands in IgG at acid conditions Meletios Roussis, Gyros We have developed a new, automated and rapid procedure for quantification of residual protein A in presence of high concentrations of IgG. A new microfluidic CD is used for analysis containing microstructures that have been equipped with a mixing chamber upstream the capture column, in which pretreatment of samples can be made prior to analysis. The sample pretreatment is performed in two steps; first the sample is treated with a first acidic buffer in order to effectively dissociate preformed complexes between IgG and protein A. Secondly, the pH of the first acid mixture is adjusted to a slightly higher but still acidic pH, at which a selected capture reagent is sufficiently active to capture free protein A in the acidified sample. The assay is finalized by adding a fluorophore labeled detecting antibody at neutral conditions. This new concept has been evaluated using using two protein A ligands and different IgG preparations to demonstrate the utility of the procedure.
Cell Culture and Upstream Processing
B1 An Integrated Approach Toward Developing a Robust VPPL Cell Line Development Platform Ying Huang, Vaccine Production Program Laboratory Accelerating timelines to deliver stable cell lines with high productivity is quite challenging, especially if biologics portfolio is quite diverse. As an integrated approach to shorten our cell line development timeline, host cells were pre-adapted in full strength production medium, several expression vector elements were evaluated and technologies like nucleofection, ClonePix, FACS and AMBR were incorporated into the process flow. Our first generation platform enabled us to generate clones expressing up to 2 g/L of antibody in shake flask culture within 4 months. To further shorten the timelines and increase throughput for clone generation we are testing several combination of selection and cloning strategies and the data will be discussed.
B2 Custom Chemically Defined Media for CAP-T Cells Gary Boch, Cevec Pharmaceuticals CAP-T Cells in Combination with a Newly Developed Chemically Defined Culture and Transfection Media - A Powerful Platform for Transient Recombinant Protein Expression
B3 DOE for Continuous BioProcessing of Therapeutic Monoclonal Antibodies Maurizio Cattaneo, BioVolutions Inc.
B4 The Effects of Prolonged Exposure to Fusible Tubing on CHO Media David Courchesne, Alexion Pharmaceuticals Fusible tubing is a standard method of aseptic fluid transfer in both upstream process development and GMP manufacturing. In this study we investigate the performance of media after varying durations of C-Flex® exposure. Two liter aliquots of media with low, moderate and high C-Flex® contact were prepared to evaluate in fed shake flasks. The performance evaluation was based on cell growth, metabolite and protein production in recombinant CHO cells. The shake flasks were performed in triplicate for each condition and sampled daily alternating each flask to preserve culture volume. The detrimental effect of fusible tubing exposure on media at high and moderate levels was immediately observed in cell growth. The condition with the highest level of exposure exhibited a significant decrease in peak VCD and two fold less productivity when compared to the control. In conclusion, fusible tubing contact should be minimized by duration and frequency of use to prevent loss of media integrity and performance. These results may not be unique to media and further investigation of fusible tubing exposure outside the scope of this study such as feeds and other additions would be prudent.
B5 A Generic Cell Growth Testing Method of Disposables: A Multi-Company Collaboration and Applications Xiao-Ping Dai, Bristol-Myers Squibb Company Disposable bags are widely used in the biotechnology industry. The two main purposes are to store cell culture media and to grow cells for inoculum or production. Several groups have reported growth inhibition resulting from the use of such products. This poster presents the outcomes from a four- company's collaboration, using several different cell lines and growth media independently, to develop a cell growth testing method. It suggests that a cell based assay is useful for troubleshooting and quality control for disposable bags for both suppliers and customers. Applications of this assay will be discussed.
B6 Development of BD Resurge™: A Family of Chemically Defined Cell Culture Formulations for Use as Bioprocess Supplements and Feeds Elizabeth Dodson, BD, Advanced Bioprocessing Optimization of cell culture parameters is an essential component of process development in biopharmaceutical industry. Every cell line offers unique challenges for developing optimal cell culture processes that yield desired growth profiles, high titers, and suitable product quality. Typically different growth conditions are attempted before process goals are met. A significant portion of the optimization process is committed to determining cell culture supplement and feed conditions that will boost performance. BD has developed BD Resurge, a diverse set of CD formulations for use in multiple cell lines and culture systems to help reduce the timelines associated with cell culture process development.
B7 Bioproduction Using Flow Electroporation Systems: Gram/L Antibody Production via Transient Gene Expression (TGE) Through Rapid, High Yield Stable Cell Line Generation Karen Donato, MaxCyte Flow electroporation technology greatly shortens the timeline of antibody development by enabling large scale transient gene expression (TGE) directly within CHO cells, thus reducing the creation of stable cells early in the development process and eliminating the need to change cell backgrounds during scale up to biomanufacturing. MaxCyte offers two flow electroporation-based systems: the MaxCyte STX® Scalable Transfection System and the MaxCyte VLX® Large Scale Transfection System. These systems offer a universal means of fully scalable, high efficiency TGE and are capable of producing multiple grams of antibodies and bi-specific antibodies following a single CHO transient transfection. We will present data demonstrating the reproducibility and scalability of MaxCyte flow electroporation as well as its capacity to produce antibody titers >1g/L using TGE. In addition, we will present data showing the use of MaxCyte electroporation for creation of stable CHO pools and the rapid generation of high-yield stable cell lines within 6-8 weeks of transfection.
B8 Linear Scalability of Virus Production in Integrity® iCELLis® Single-Use, Fixed-Bed Bioreactors From Bench Scale to Industrial Scale Stéphanie Dubois, ATMI LifeSciences Viral vaccines are usually produced by anchorage-dependent cells in static multitray systems, roller bottles or bioreactors with microbeads. However, these technologies do not enable process intensification as they involve many manual operations. To enable process intensification, ATMI developed iCELLis®, a scalable range of disposable pre-packed fixed-bed bioreactors that operate in perfusion mode. The fixed-bed accommodates up to 500m² of growth surface area in only 25 liters reactor volume. It can be inoculated at a very low cell density which simplifies seed train. Here we present the scale-up of MDBK, Vero and HEK293 processes from iCELLis benchtop bioreactor to production units. Scaling-up with iCELLis systems is quick and similar to that of chromatography columns. As the bioreactor scale increases, the fixed-bed height remains constant while the diameter increases. Small scale processes were transferred to large scale iCELLis by keeping same culture parameters (pH, DO & Temp), identical ratios for cells/surface and media/cells, compaction of carriers, the linear speed through the fixed-bed and the perfusion rate. The study demonstrated that iCELLis bioreactors simplify viral production processes and that linear scale-up in iCELLis is easily developed from 0.53m² to 500m² in less than one year.
B9 Transferring an Upstream Process for a Therapeutic Glycoprotein to a Commercial Facility John Facenda, Alexion Pharmaceuticals, Inc Extensive work was recently done to develop the CHO cell culture process for an enzyme replacement therapy. With the completion of clinical batches, the process is now being run at commercial (10,000L) scale. In advance of process transfer to the commercial facility, a collaborative effort between manufacturing and process development staff identified several gaps in the facility's ability to run the process. Gaps related mostly to equipment capabilities and included the quantity and capacity of support vessels, oxygen transfer limitations and addition tube configuration. Each of these fit issues was known to be essential for maintaining productivity and/or product quality. The solutions to address them involved add-on modifications that could be completely removed following the campaign to leave the facility and its equipment exactly as found. It was a mandate that any changes did not interfere with the validated facility's ability to produce an approved drug. Additional discussions between manufacturing and process development found other process fit issues not initially discovered. The ability to regulate the amount of inoculum transferred, for example, was found to be unfeasible with existing automation. This directly impacted the ability to achieve a specific seeding density, one parameter that a number of other key parameters depend on. In addition to addressing facility fit issues, process development focused on meeting several process improvement challenges identified during clinical production. The upstream process for this glycoprotein is rather complex compared to the monoclonal antibody process the commercial facility is accustomed to performing. This presentation will focus on the solutions identified to resolve facility fit issues, as well as streamline process intricacies, resulting in the successful transfer of a pipeline process to a manufacturing facility.
B10 Assessment Of Process Performance And Product Quality In High Performing Fed-Batch Cultures Thomas Falkman, Ge Healthcare Higher yields, more potent compounds, smaller batch sizes and the cost pressure on R&D budgets push the production of biopharmaceuticals towards single-use bioreactors. Simple cultivation systems can be an attractive platform for the production of e.g. monoclonal antibodies by high cell density fed-batch processes. We have developed a high cell density fed-batch process based on an IgG1 producing CHO cell line and the ActiCHO Media System platform in stirred tank bioreactors as well as in WAVE Bioreactor system. Furthermore, the product quality for key product quality attributes, i.e. glycan distribution, molecular size distribution and charge heterogeneity was analyzed.
B11 Three Bioreactor Configurations for Z®RP Cell Culture System Stanley Goldbeg, GLEN MILLS INC. The Z®RP Cell Culture System has introduced three configurations of the core Bioreactor. Each design can accelerate the expansion of specific cell lines while maintaining homogeneous culture for weeks on end. Optional sizes for each bioreactor model allow for specific cell densities when scaling to greater numbers. Data for numerous cell lines, such as Natural Killer (NK) cells, is presented
B12 Influence of Soy Protein Hydrolysates on Robustness of Cell Culture Experiments Kathleen Harrison, Frieslandcampina Domo Hydrolysates are complex media supplements composed of many compounds. In numerous studies till date, the beneficial effect of supplementing hydrolysates in cell culture applications has been shown. These proven benefits are primarily related to enhancement in cell growth and recombinant protein production in cell culture applications. In addition to these benefits, in this poster, for the first time some remarkable results in relation to the role of Proyield Soy SE50MAF-UF in improving the reproducibility of small scale cell culture assays are represented. More specifically, as compared to the chemically defined media, the addition of Proyield Soy SE50MAF-UF significantly reduced the susceptibility of CHO cells for temperature fluctuations during the cell culture assay.
B13 TAPBOOST technology: Novel Technology to Enhance the Production of Therapeutic Recombinant Proteins Through Protein Folding System Akinori Hishiya, Boston Strategics Corporation Although protein folding is very important for the production of therapeutic proteins, due to the complicated protein folding system, the technology to specifically promote protein folding of targeted proteins has not been successful yet. TAPBOOST technology is a novel technology which exploits the cellular protein folding system. In TAPBOOST technology, a proprietary protein called "TAPBOOSTER" is expressed together with the therapeutic protein. Selective TAPBOOSTER can be designed to target any kind of protein. The TAPBOOSTER targeted protein is 1) functional, 2) stable, and 3) easy to secrete. All these effects significantly enhance the production of the targeted therapeutic protein. TAPBOOSTER has successfully enhanced the production of many therapeutic recombinant proteins such as Humira® (9 folds), DP12® anti-IL8 antibody (8 folds), Enbrel® (6 folds), and Avastin® (4 folds). Importantly, TAPBOOST technology is very effective especially to proteins with protein folding problem such as IL13Ralpha2-Fc (Fc fusion protein with IL13 receptor alpha2 truncated form). By using TAPBOOST technology, the productivity of IL13Ralpha2-Fc was drastically improved more than 20 folds. The unique mechanism of TAPBOOST technology enables this technology to combine with any other existing technologies to enhance the production of therapeutic protein with synergistic effect.
B14 An Integrated Approach Toward Developing a Robust CHO Cell Line Development Platform Ying Huang, National Institutes of Health Accelerating timelines to deliver stable cell lines with high productivity is quite challenging, especially if biologics portfolio is quite diverse. As an integrated approach to shorten our cell line development timeline, host cells were pre-adapted in full strength production medium, several expression vector elements were evaluated and technologies like nucleofection, ClonePix, FACS and AMBR were incorporated into the process flow. Our first generation platform enabled us to generate clones expressing up to 2 g/L of antibody in shake flask culture within 4 months. To further shorten the timelines and increase throughput for clone generation we are testing several combination of selection and cloning strategies and the data will be discussed.
B15 Evaluation of Adventitious Agent Barriers to Leptospira sp.: A Novel Bacterial Contamination in Cell Culture Manufacturing Ting-Kuo Huang, Genentech, Inc A novel bacterial contamination was observed in a CHO cell culture GMP manufacturing process during routine microscope visual examination of the cell culture samples and was identified as Leptospira licerasiae. This is a novel organism contamination issue, to our knowledge, in the whole biological production network. Neither cell culture performance (i.e. pH, dO2 and cell culture performance) nor current QC standard confirmatory contamination testing can detect this bacterium. To provide a more robust microbial control, three different adventitious agent barriers, including filtration, UV and HTST (High Temperature Short Time) technologies, were evaluated based on their barrier effectiveness in terms of 1) no Leptospira growth is recovered after barrier treatment, and 2) no significant barrier impact on cell culture performance and media quality. HTST is recommended to be the most effective barrier against Leptospira. The impact of HTST on commercial CHO cell culture media is further evaluated in terms of cell culture performance. This presentation highlights the key facts and important findings which are valuable for other partners in cell culture manufacturing network
B16 Development of a Perfusion-based Non-Centrifugal High Density Cell Banking Platform Xiaoxia Jin, Genzyme, a Sanofi Company In order to shorten and simplify the current upstream seed train culture expansion operations, a perfusion-based, non-centrifugal high density (HD) cell banking platform was developed. Various cell retention methods including alternating tangential flow (ATF, by Refine Technologies), tangential flow filtration (TFF), and built-in floating filter were evaluated. ATF4 was selected because of its superior performance in supporting a HD perfusion culture in a 10-L Wave Cellbag bioreactor as well as a fast in-vessel cell concentration. A simplified process control strategy was also developed so that desired pH and DO ranges can be maintained by manually adjusting gas compositions and rocking conditions. This strategy eliminated the dependence on online pH and DO feedback controls. When the culture reached a viable cell density of > 3×10^7/mL, it was concentrated to > 11×10^7/mL, and was then frozen at 10×10^7/mL in 5-mL cryovials. It has been demonstrated that one HD vial enables the direct inoculation of a 1 L culture with high growth rate and viability. This HD banking platform has been successfully tested with three CHO cell lines expressing different recombinant proteins/antibodies.
B17 Achieving high titers using CHO cells to express proteins in a single medium for growth and transient transfection in batch culture Kalle Johnson, Thermo Fisher Scientific Transient gene expression (TGE) is a widely used technique to rapidly produce recombinant proteins for evaluating various attributes including structural and functional characterization during preclinical studies. Although stable Chinese hamster ovary (CHO) transfectants are routinely used for large-scale production of recombinant protein, TGE in CHO cells has historically provided considerably lower amounts of recombinant material for evaluation. This has prompted the use of TGE in higher yielding human embryonic kidney (HEK293) cells to evaluate target proteins before stable CHO transfectants are made. Unfortunately, differences in the metabolic pathways between CHO and HEK293 cell lines can lead to differences in the characteristics of the proteins produced in the two cell types. The use of multiple media during TGE, such as one media for transient transfection and another media for production, makes the process cumbersome. These experiments show that transient work can be completed with minimal media changes in the same CHO line as for stable production. This new formulation provides high transfection efficiencies and yields of recombinant protein during TGE batch cultures of CHO cells in a single serum-free medium for transfection, growth and production.
B18 Viral Vector Production in the Integrity® iCELLis® Single-Use Fixed-Bed Bioreactor, From Bench Scale to Industrial Scale Matthew Kremer, ATMI LifeSciences Background and novelty Recombinant viruses (e.g. lentivirus, AAV) can be used as human gene therapy vectors. They are produced in adherent cell cultures using transient transfection or infection strategies. Therefore, iCELLis® bioreactor (ATMI LifeSciences) offers a new alternative with stronger process controls and ease of scale-up. It is designed for adherent cell growth on microfibers carriers packed in a fixed-bed. Experimental approach A mirrored approach of the previous production in static vessels was set-up in the small scale iCELLis bioreactor. Transfection/infection efficiency in the system was evaluated by Flow Cytometry (measuring the expression of protein marker). At harvest, extracellular viral vectors are collected in the medium while intracellular vectors harvest requires cell disruption directly in the bioreactor by physico-chemical methods. Results and discussion We present results of AAV and paramyxovirus production in the iCELLis nano versus classical culture recipients. These results indicated higher titer for AAV (5.108 vg/cm²) in small scale iCELLis versus 3.108 in Cells Stack-5 plates and 0.5-1 log higher titers than control for paramyxovirus. Results of transient transfection processes by PEI showed similar transfection levels in iCELLis than in CF. Results in iCELLis demonstrated good virus productivity with identic infectious units for viral genomes than in control.
B19 Orbital Mixing as an Alternative to Stirred-Tank Bioreactors for Cell Culture Scale-Up and Commercial Production David Laidlaw, Kuhner Shaker Inc Commercial-scale production of therapies requires scale-up from small scale to large scale for delivery of product to market. Here, orbital shaken bioreactors are presented as an alternative to stirred-tank vessels for process development and scale-up. Orbital shaken bioreactors offer a low shear and technically conservative approach capable of preserving mixing hydrodynamics from the µl stage to scales as large as 2500L. The speed of scale-up is fast and the cost of implementation is low in comparison to stirred systems. kLa's and mixing times for scales from 5ml to 2500L are presented here.
B20 Is Your E. coli Cell Bank in Control? Debbie Letham, Charles River Laboratories E. coli cell lines are often utilized as hosts to express recombinant genes. Common strains utilized in the biopharmaceutical industry are designed to achieve high-level expression of heterologous proteins from recombinant vectors. Unfortunately, the design of certain cell lines, including BL21 DE3, included insertion of bacteriophage lambda DE3 into the host strains. This phage encodes a T7 RNA polymerase that functions to drive protein expression from plasmids containing a T7 promoter (e.g., pET vectors). Since GMP regulations require cell line characterization of cells used to produce biological materials, biopharmaceutical companies need to know that their protein-producing cell lines are not unstable due to loss of the lambda DE3 prophage (integrated, lysogenic bacteriophage), which may also lead to cell lysis. This would indicate that their system is not in control. In addition, contract manufacturing facilities need to know if a potential phage infection from one client's cells will contaminate future non-lysogenic strains. While bacteriophage replication will lyse cultures of bacteria rapidly, lysogenic cell lines may grow without cell lysis for many generations with little spontaneous prophage induction. Lambdoid prophage induction can be stimulated by factors that initiate the SOS stress response, possibly under certain conditions found in bioreactors. Test methods to detect latent bacteriophage infection expose cell lines to conditions or reagents that may trigger prophage induction (e.g., mitomycin C). Results may include reduction in the turbidity of a liquid culture and the generation of plaques when cultures are mixed with permissive bacteria on agar plates. While lambda DE3 lysogens were designed to have limited ability to excise, induction triggers, as well as spontaneous induction, have led to the observation of lysed cultures and phage particles during E. coli cell bank characterization studies. In this presentation we discuss the potential of cell lines containing lambda DE3 to become lytic during bacteriophage screening with the potential to cause delays in testing and production schedules. We describe gel-based and fluorescent PCR assays as methods to screen E. coli cell cultures and phage plaques for lambda DE3 sequences, and a method to differentiate between wild type lambda phage and lambda DE3 phage.
B21 Microbial Cultivation in Different Scales in the CELL-tainer® Wave-mixed Single-use Bioreactor Lexan Lhu, Charter Medical Single-use bioreactors are applied in the biopharmaceutical industry for mammalian cell culture processes. For microbial processes, the CELL-tainer® technology allows gas-liquid mass transfer rates comparable to stirred tank reactors. In the CELL-tainer, the rocking motion of the bag is generated with a combination of a 2-D rocking motion so the turbulence in the liquid is intensified. Thus, volumetric oxygen transfer rates (kLa) of over 400 h-1 could be achieved. The scale-up of E.coli nutrient-limited fed-batch cultivation from 15L to 150L scale in the CELL-tainer single-use bioreactor has been conducted. A final biomass concentration of 45 gL-1 within 24 hrs was obtained proving the suitability of this reactor for the application of bacterial processes. A good process performance has also been obtained with marine, shear-sensitive heterotrophic algae in the CELL-tainer, proving the suitability for this kind of bioprocesses which the application of stainless steel-based reactors is restricted due to corrosion. The combination of intelligent software sensor control and improving sensors will lead to improved control of bacterial fed-batch processes. The availability of single-use bioreactors for microbial cultivations widens their potential, not only in biopharmaceutical processing, but also as a pre-culture bioreactor for large processes and as a tool in bioprocess development.
B22 Mgat4 May Play a Role in Increased Sialylation by Overexpressing Functional MGAT1 in Mgat1-Disrupted Chinese Hamster Ovary (CHO) Cells Nan Lin, Sigma-Aldrich/SAFC MGAT1 adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) structure. Goh et al. reported increased sialylation after restoring MGAT1 function in MGAT1 deficient CHO cells. The hypothesis is that Mgat1 disruption and restoration may lead to altered N-glycan biosynthesis gene expression, which contributes to increased terminal sialylation. Zinc-finger nuclease (ZFN) technology was used to create Mgat1-disrupted CHO lines with high-mannose dominant simple N-glycoforms. A functional hamster MGAT1 was overexpressed in an Mgat1-disrupted IgG producing cell lines (Mgat1 KO37). Five out of sixteen clones isolated from the MGAT1 KO37 OE stable pool demonstrated restored complex N-glycoforms, and two demonstrated 1.4-fold higher sialic acid compared to the non-ZFN transfected line. All Mgat1 KO37 OE clones showed significantly increased Mgat1 expression by q-RT PCR than the endogenous level in the non-ZFN transfected line. The host cell line (CHOZN® GS) had significantly higher endogenous Mgat1 expression than the IgG expressing cell line. Mild increase of the branching enzyme Mgat4 was observed in one of the clones with increased sialylation. HPAEC-PAD analysis was used to analyze the content of tri- and tetra-sialylated glycans that are enzymatic products of MGAT4. The results support co-overexpression of Mgat1 and Mgat4 to create host cell lines for biopharmaceutical production with increased sialylation.
B23 Scale-up studies in single-use environment to achieve high cell density cultures Ben Madsen, Thermo Fisher Scientific Scaling up from bench-top bioreactors to large volume perfusion cultures has proven difficult from the standpoint of both bioreactor and perfusion technologies. Studies were undertaken with single-use hollow fiber perfusion technologies and single-use bioreactors to determine the effects membrane chemistry, pump strategy, and filter surface area have on achievable viable cell density, protein production, and protein harvest. Using a 50 liter bioreactor, viable cell densities of 1.22 x 108 cells/milliliter and bioreactor protein concentrations of 1.15 miligrams/milliliter were achieved. It was also shown that a large filter surface area to bioreactor volume ratio can lead to significant protein loss. Additionally, sparge strategies and bioreactor controls need to be adapted to the various demands from high-cell density cultures. The data presented here suggest that scale-up to larger-volume perfusion cultures is achievable with robust process development activities focused on the unique aspects of perfusion, bioreactor, and control technologies.
B24 Development and Characterization of a LONG®R3 IGF-1 ELISA Kit Ariane Marolewski, Repligen Corporation LONG®R3 IGF-1 is an analog of IGF-1 which has been engineered for improved bioavailability compared to IGF-1. It binds to and activates the Type I IGF-1 receptor, leading to improved cell growth and productivity. Quantitation of LONG®R3 IGF-1 is important when developing cell culture processes in order to optimize the concentration of LONG®R3 IGF-1 used and feeding strategies. Quantitation is also important when developing a purification process, in order to demonstrate clearance. An ELISA has been developed for the quantitation of LONG®R3 IGF-1 in media and drug substance samples. The assay uses a sandwich ELISA format with colorimetric detection. The performance characteristics of the assay will be presented including accuracy, precision, linearity, LOQ, recovery from media, and range.
B25 Signal Peptide Optimization: Effect on Recombinant Monoclonal IgG Productivity, Product Quality and Antigen-Binding Affinity Joaquina Mascarenhas, SAFC Sigma Aldrich A signal peptide is a 5-30 aa peptide present at the N-terminus of secretory proteins. Signal peptides are known to have a strong impact on both the efficiency of protein secretion and correct processing at the N-terminus. N-terminal heterogeneity can have an impact on both the product quality and bioactivity of the bio-therapeutic product. In this study, we evaluated two signal peptide sequences for a model recombinant humanized IgG. Productivity was evaluated at the transient transfection and stable (bulk and minipool) stage using FACS analysis. N-terminal cleavage was evaluated using peptide mapping and intact mass analysis. N-terminal heterogeneity was observed with one of the signal peptides resulting in a major species with delta mass 159 added to the N-termini of the Light Chain. No effect on either the product quality or the antigen binding affinity of the purified product was seen. Site directed mutagenesis was successfully used to re-engineer the signal peptide to prevent this mis-cleavage. However, this resulted in a decrease in the productivity of the recombinant IgG, without affecting the product quality or bioactivity. The results underscore the importance of evaluating more signal peptide designs with a balanced optimization strategy to ensure correct N-terminal cleavage and high productivity
B26 Optimizing a single serum-free medium for high density growth and high efficiency transient transfection of multiple HEK293 cell lines using conventional reagents and methods Gerald McEwen, Thermo Fisher Scientific Frequently in the biopharmaceutical industry, transient gene expression (TGE) is used to produce recombinant proteins for purposes such as biophysical characterization and clinical evaluation. These techniques require the rapid introduction of the gene of interest into human embryonic kidney (HEK293) cells. Often this is done by complexing the recombinant DNA with a transfection reagent. The complexing process and subsequent introduction into the cells is frequently inhibited or extinguished by media components, which has led to the use of very lean serum-free media for transfection and different media for production. Here we describe the use of design of experiments (DOE) mixture design and high throughput screening (HTS) techniques allowing for the development of a complete, serum-free medium formulation intended for both growth and transient transfection. Additionally, very high transfection efficiencies and protein titers were achieved in the formulation while using conventional transfection reagents and techniques such as 25 kilodalton linear PEI and liposome based transfection methods.
B27 Automated On-line Sampling and Nutrient Monitoring for Characterizing Glucose Consumption of Microbial Fermentation Cultures William Miller, Flownamics A Seg-Flow® automated on-line vessel sampling system (Seg-Flow System) was evaluated for characterizing real-time glucose consumption of microbial fermentation cultures used for protein biotherapeutic production. High cell density fermentation processes employing host strains of Scarab Genomics' Clean Genome® E. coli were conducted at 10L working volume using a biphasic approach. Cultures were grown to a specific density in minimal salts/glucose media during the batch phase. Upon reaching the targeted density, glucose was fed exponentially to control the culture growth rate at a specified constant µ hr-1during the fed-batch phase. The Seg-Flow System, consisting of the Seg-Flow autosampling module, a YSI® Biochemistry Analyzer and a fraction collector, provided real-time analysis of media glucose concentrations as well as programmed time-point sampling for manual glucose analysis and additional off-line analytics. Automated and manual glucose analyses were comparable, which demonstrated fidelity of the results generated by the Seg-Flow System. The Seg-Flow System provided an automated, real-time solution for monitoring glucose concentrations and, in turn, characterizing glucose consumption of E. coli fermentation cultures.
B28 Scale-Up of Hepatic Progenitor Cells from Multiple Tray Stack to 2-D Bioreactors Fabien Moncaubeig, ATMI LifeSciences Cell therapy is offering a promising future in medical advances. While multilayer trays for cell amplification are suitable for R&D and preclinical purposes, they cannot support the large-scale industrial production. Xpansion™ Multiplate Bioreactors have been designed to enable easy transfer from existing multiple-tray-stack processes by offering the same cell growth environment on 2D hydrophylized Polystyrene (PS) plates in a compact and closed system. Preserving the cell culture environment is critical as small variations in physicochemical parameters can heavily impact cell growth and behavior. The Xpansion-10 plates (0.6 m²) is used first as a scale-down model to screen critical parameters and best conditions before scaling up the process. Next, scale-up was assessed on Xpansion-50 plates (3.05m²) before achieving the final production scale using Xpansion-200 plates (12.2m²). Data showed a similar QC results and cell growth at all scales of the Xpansion systems compared to the Cellstack (Corning) control. Final scale Xpansion-200 plates lead to a production of ~3.0x109 cells. Process transfer from Cellstack to the Xpansion platform was successful. Cells retained their therapeutic potency. Reproducibility and reliability, as well as the feasibility of using Xpansion bioreactors as a tool for cell amplification, were demonstrated.
B29 Optimization of CellventoTM CHO-200 Production Media and Feed Performance Through Experimental Evaluation of Multiple Fed-Batch Processes John Mumira, EMD Millipore The purpose of a fed-batch process in cell culture is two-fold; first, a production medium is used to support initial cell growth and production, and secondly, a feed medium is added to replenish depleted nutrients required for cellular function and to maintain the production phase of the culture. Since production media and their companion feed(s) are typically dependent on each other, optimizing a feeding strategy is crucial to achieve a culture that both grows and produces effectively. While recommended ranges of feed volumes and frequencies are typically provided with the products, optimal feed conditions typically need to be empirically determined for a given cell line and its associated process.
B30 Building Efficient Protein Biologics Workflows Using Microchip Analysis Platforms Tal Murthy, PerkinElmer Proteins biologics is an important aspect in the life-sciences biotech and pharmaceutical industry. Advances in automated procedures facilitated production of proteins and rapid analytical characterization. Almost all methods of analyses rely on accurate and rapid characterization of proteins before subjecting to downstream assays. Traditionally, proteins were analyzed using a spectrometer or colorimeter reagent based methods for total quantification, and gel based analysis for detailed sizing information. Although widely accepted and economical in lower throughput environments, these techniques suffer from some disadvantages especially when sample numbers increase. For example, classical spectrometric and gel based analyses requires large amounts of protein sample, may not have the dynamic range, involves tedious procedures and are not suitable for processing large numbers of samples. Commercial organizations came up with some improvements to address the limitations in rapid high-throughput analysis and characterization. One such promising technology is the use of microchip platforms for analysis. The microchips require low amounts of sample, offer a wide dynamic range and are readily suitable for analysis of several samples in a fraction of the time. In this study we report two microchip platforms for assessing total and detailed analysis of proteins. Some results from these platforms are presented. Efficient use of either or both of these platforms in high-throughput workflows will permit accurate analysis of proteins and reliable output.
B31 Developing a Scalable, High Performance Bio-Production Process in Minimal Time Thomas O'Brien, Becton Dickinson With the tight timelines associated with the biotherapeutic product development and launch process, process development groups are challenged with developing robust, scalable processes in minimal time. Cell culture media and feeding strategies are critical factors, so it is essential to quickly identify a high performance process that is easily adaptable and scalable. One approach is to quickly screen commercially available culture media and feeds to identify those appropriate for the production process. However, this approach provides little, if any, insight into the formulations and key performance drivers, making it difficult to apply to a platform process. An alternative strategy is to perform media optimization and feed studies to design a process that can be cell line specific or applicable to a production platform. Through the application of appropriate expertise and high throughput technologies, media design and optimization can offer a rapid, effective solution to biotherapeutic development challenges. A series of studies were performed using scale-down models to quickly identify a base medium that demonstrated high performance with CHO lines. The evaluation included the use of Duetz shaking multi-well plate system for media screening and optimization studies. The ambr® microbioreactor system was used to model the conditions that would be observed in a scaled up bioreactor system. Feed studies were also conducted using the ambr® system to further enhance the performance of the base medium. By using predictive scaled down screening methods, a large number of conditions were rapidly screened using minimal resources. As a result, an optimized base medium and feed were quickly identified as high performing candidates for use in a scaled up process.
B32 Comparative and Scale-Up Studies of BD Resurge™: A Set of CD Cell Culture Formulations for Use as Feeds and Supplements in Bioproduction Duncan Omune, BD, Advanced Bioprocessing During biopharmaceutical process development, one challenge facing process development scientists is the initial selection of appropriate cell culture supplements or feeds that will give desired critical performance attributes when used in large scale bioproduction systems. Availability of a family of chemically defined (CD) supplements offering diverse performance profiles can significantly improve the chances of finding an effective supplement or feed. We have developed BD ResurgeTM CD PAK to address these issues of feed/supplement selection and scalability. The current studies were performed to compare BD Resurge CD formulations to commercially available CD feeds/supplements and to evaluate their use at larger scales.
B33 Optimization of Cell Banking Parameters for a Temperature Sensitive Cell Line Douglas Osborne, Biogen Idec During generation of a working cell bank (WCB), an approximate 10% drop in culture viability was experienced following culture centrifugation and immediately prior to vial dispensing. Following completion of cell banking activities, vials were thawed demonstrating poor recovery in both cell growth and viability. An extensive cell banking characterization was carried out to identify root cause for the drop in culture viability during the cell banking culture pooling step and also optimize robustness of the cell banking process. Results from the characterization identified processing time, culture temperature, freezing equipment, and sensitivity to shear as parameters for optimization of the cell banking process.
B34 Accelerating Time to Market for Proprietary Cell Culture Products by Leveraging Statistical Design of Experiments and High Throughput Liquid Handlers Sonal Patel, EMD Millipore Chemically-defined cell culture media and feed formulations can contain 50-70 individual raw materials. These formulations are largely based on historical precedence and classical serum-supplemented media that were developed to support anchorage dependent and batch cell culture applications. Cell culture media and supplements supporting CHO suspension cell lines and fed-batch manufacturing applications require significant optimization, including performance validation with multiple cell lines and scalability testing. Typically large, inefficient experiments are utilized to screen and identify factors and concentrations that provide a positive effect on the cellular metabolism and antibody production. In order to accelerate developmental timelines and expedite the simultaneous screening of multiple components or factors, while reducing the total labor and lab resources required and minimizing the process scale, the Cell Culture Development Team at EMD Millipore has successfully implemented high throughput approaches for basal media and feed supplement development. We have completed development of chemically-defined media and feed formulations for use with CHO DG44 cell lines using high-throughput cell culture screening platforms, automated liquid handlers and statistical design of experiments.
B35 Leveraging the Modular, Automated Sampling Technology (MAST) Platform for Merging In-Line and At-Line Analytical Technologies To Gain Optimized Cell-Level "Observability" and Data-Driven Process "Guidance" Clint Pepper, Bend Research 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 online tools like dielectric spectroscopy and Raman are helping to provide insight, there is still a gap integrating the data produced by these techniques with offline measurements such as cell density, viability, metabolite levels, and titer. Bend Research Inc., in collaboration with Pfizer Inc. and other major biopharmaceutical companies, is working to advance the Modular, Automated Sampling Technology (MAST) platform, which provides samples directly from bioreactors to analytical devices while maintaining process sterility. Early studies with this modular sampling platform have demonstrated that automated at-line measurements are representative of parallel manual samples. During these studies, maintenance of sterility has not been an issue. This poster illustrates how the MAST platform is incorporated with on-line analytical technologies to deliver overall cell-level "observability". For example, as a result of this integration, dielectric spectroscopy measurements can be corrected to more accurately estimate total and viable cell density. 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.
B36 LONG®R3 IGF-1 Supplementation Results in Improved CHO Productivity versus Insulin in Modern CHO Fed-batch Media Jinghui Qian, Repligen LONG®R3 IGF-1 is a human IGF-1 analog containing a 13 amino acid N-terminal extension and a mutation at position 3. It activates the Type 1 IGF receptor, which is responsible for growth-promoting and protein synthesis effects in CHO cells. A common growth factor supplement used in CHO media, insulin, acts primarily through the IGF-R. LONG®R3 IGF-1 has been shown previously to increase CHO productivity. However, previous characterization studies were older and used primarily DMEM/F12 media. As serum-free media development has progressed, media have become more complex and richer. To establish the effect of LONG®R3 IGF-1 in media that meet today's industry standards, the effect of LONG®R3 IGF-1 on the productivity of antibody-expressing cell lines was compared to that of insulin and no growth factor supplemented media. Media with LONG®R3 IGF-1 gave superior titers versus no growth factor or insulin in several instances. In addition, it was demonstrated that a combination of LONG®R3 IGF-1 and Transferrin gave higher titers than media supplemented with commercial ITS (insulin, transferrin, selenium). LONG®R3 IGF-1 is used at low concentrations, 10 - 100 ng/mL, in cell culture and is already used in several commercial antibody manufacturing processes. Therefore, it represents a regulatory-friendly method of enhancing serum-free cell culture performance. LONG®R3 IGF-1 should therefore be considered as a media supplement to further optimize a protein-free process or to replace insulin in existing processes.
B37 Universal, Fully Scalable Transfection Platform for Production of Complex or Difficult-to-Express Proteins: Superior Performance over Other Transfection Methods & Expression Systems Mark Schmeizl, MaxCyte Biotherapeutic development often requires the production of gram level quantities of recombinant proteins. Transient transfection offers a means of rapidly expressing a variety of proteins including antibodies, antibody-like molecules, biochemical targets of interest, vaccines, viral vectors and virus-like particles (VLPs). Although a variety of transient transfection methods are available, most do not meet the requirements of scalability, consistency and cell type flexibility. MaxCyte's proprietary flow electroporation technology produces proteins from a variety of adherent and suspension cell types faster than creation of stable cell lines. In this poster we describe large scale electroporation using the MaxCyte STX Transfection System for the production of several antibodies, including bi-specific antibodies, VLPs and a lentiviral vector. Data will be presented for high efficiency transfection of cells commonly used in protein production including CHO, HEK293 and insect cells. Results from comparisons to other transient transfection technologies such as lipid reagents and PEI demonstrate the superior utility and quality of MaxCyte electroporation.
B38 Strategies for Investigation of Cell Culture Media Failures Kim Schrag, SAFC Troubleshooting performance issues with cell culture media is complicated by many variables including undefined materials, chemical interactions and raw material variation. Consequently, a systematic approach is required for investigating cell culture media failures. Following verification of the failure, the first step in the investigation process is to identify a control lot (a lot that performs as expected during quality testing) for comparison to the medium product in question. Subsequently, a review and comparison of the batch records, packaging, storage history, and use of the material is performed. However, identifying root cause of a medium failure may require more extensive investigation and is facilitated by multivariate analysis (MVA). Recently, three lots of insect cell culture media, EX-CELL™ 420, which contains 50+ raw materials, failed growth promotion testing with Sf9 cells. An internal investigation into the failures was initiated and consisted of a systematic approach utilizing risk assessments and MVA for identification of suspect raw materials. A fractional factorial DOE confirmed the root cause as variability in yeastolate used to formulate the medium. This has resulted in development of an improved performance-based growth promotion assay for screening in-coming yeastolate lots as a corrective action to improve consistency of medium performance
B39 The effect of copper sulfate on CHO cell culture processes and lactate metabolism Rochelle Shapland, Alexion Pharmaceuticals The use of copper sulfate in mammalian cell culture processes has proven to be beneficial for many CHO cell culture processes. We have developed a copper sulfate containing platform process using mAb-producing and non-mAb producing CHO cell lines. An important benefit through the supplementation of copper sulfate is increased lactate consumption during a fed batch cell culture process. We have found that lactate producing cell lines in the absence of copper sulfate shifted to lactate consuming phenotype in the presence of copper sulfate. While the exact mechanism of copper sulfate in cell culture processes is unknown, our studies suggest that copper sulfate enhances the oxidative metabolic capacity of cells. This was supported by testing of cell culture pellets and supernatants for by-products of glycolysis, the TCA cycle, and the PPP. The effect of copper sulfate was found to be scale-independent. Increased lactate consumption and decreased base use for pH control translated to improved process scalability. Our studies were conducted in shake flask, as well as 2-10L, 45L, and 200L bioreactors.
B40 Improvements to cell line screening and process development through automation methods Jennifer Smith, MEDIMMUNE A retrospective data review of upstream process development projects producing monoclonal antibodies (mAbs) from recombinant CHO cell lines for early phase clinical trials was performed. This identified opportunities to accelerate timelines through efficiency savings and the introduction of high throughput automation, namely ambr (automated microbioreactor system). As a result, phenotypic stability has been taken off the critical path from lead clone selection and these activities combined with process development using a new streamlined upstream strategy for mAb projects for early phase clinical evaluation. The new approach reduces the number of clones assessed for phenotypic stability and combines experimental stages using high throughput automation, wherever possible replacing shake flasks and 5L bioreactors with ambr runs. Significant time savings of 8-10 weeks have been achieved through improvements to the cell line screening process and with the adoption of the new streamlined upstream process development approach through the introduction of high throughput automation. This strategy also has the potential to reduce the amount of process development required to make the process ready for later phase clinical evaluation.
B41 Method For Scaling-up and Producing Adenoviral and Lentiviral Particles in the Corning® HYPERStack® Cell Culture Vessel Katherine Strathearn, Corning Life Sciences "Viral transductions are used at the preclinical and clinical stages in the vaccine industry leading to an increase in demand to produce more virus more efficiently. The Corning® HYPER technology and microcarrier beads offer the ability to increase efficiency by increasing surface area without increasing spatial footprint. The focus of this study was (i) to evaluate the use of the Enhanced Attachment microcarrier beads on cell lines typically used in the vaccine-producing industry (e.g. Vero, MRC-5, and/or HEK-293AD) and (ii) to determine the efficacy of generating Adeno- and lentiviral particles using the unique Corning HYPER technology. To optimize cell scale-up on the Corning microcarrier beads various conditions were evaluated using multiple vaccine producing cell types. The data presented here demonstrate the optimal protocol for cell growth and demonstrate similar cell expansion compared to equivalent technologies. To assess viral production on the HYPER technology HEK-293AD (adenovirus) or HEK-293LTV (lentivirus) were transduced (adeno) or transfected (lenti) on the HYPERStack-12 to produce the viral particles. The results demonstrated that adeno- and lenti-viral particles can be generated in the Corning HYPERStack vessel at similar titers compared to traditional tissue culture vessels, while allowing for greater virus production in a smaller footprint. "
B42 Effects of Lowering Culture Temperature in a Continuous Perfusion Platform for Optimization of Upstream Bioprocessing Nicholas Sullivan, Gallus BioPharmaceuticals Upstream bioprocess development involves characterization of process parameters and their impact on cell growth and productivity. Process variables will differ based on the cell line and molecule under study. In addition, optimal settings may vary based on the cell culture platform utilized. While fed-batch culture has become quite popular, continuous perfusion systems offer strategically advantageous benefits for the production of unstable or complex recombinant proteins. It is, therefore, necessary to recognize the fundamentally different effects that varying process parameters can have based on the cell culture platform used. Reduced temperature, for example, has been shown to stimulate increased productivity in batch and fed-batch culture and, while this phenomenon is frequently exploited in current bioprocesses to optimize productivity, the implementation of a temperature reduction strategy has the potential to upset the somewhat delicate process of continuous perfusion. Here, we examine the complex role of temperature during the process optimization of a disposable CHO cell perfusion platform utilizing an Alternating Tangential Flow filtration system. The effects of lower temperatures on growth rate, purge rate, and filter retention are explored as well as strategies for identifying optimal temperatures to maximize the volumetric productivity and overall value of an upstream bioprocess
B43 Corynex Expression Technology: Effective protein secretion platform for "difficult-to-express" proteins Yasuhiro Takenaka, Ajinomoto Althea Inc Corynex® is a microbial protein secretion system using Corynebacterium glutamicum, a gram-positive non-sporulating bacterium, which has been used and certified as safe for the industrial production of amino acids in food additives and pharmaceutical products for several decades. Advantages of the Corynex® system enable complex human proteins including disulfide bonds, homo-dimer or hetero-dimer structures to be secreted into growth medium as active form with high purity. Newly identified Tat-pathway, which differs from the general protein-secretion pathway well known as Sec-pathway, has dramatically enhanced versatility and success rate of Corynex® for the secretion of difficult-to-express proteins. Ajinomoto Althea Inc., a contract development and manufacturing organization in San Diego, has perfected protein expression service that can reduce production costs and speed time to market - together with its cGMP compliant biologics manufacturing facilities up to 1,000L scale, to whom having troubles in protein expression or refolding.
B44 Evaluation of Microcarrier-based Suspension Cultures for Human Mesenchymal Stem Cells Jennifer Weber, Corning Incorporated A microcarrier-based suspension culture was explored for scale-up of human mesenchymal stem cell (hMSC) expansion in xeno-free medium using synthetic peptide acrylate surface beads. Cells were maintained on Corning® Synthemax® II polystyrene and CELLstart™-coated Solohill plastic microcarriers  for 14 days in xeno-free medium. In separate experiments, bone marrow (BM) derived- and adipose tissue (AT) derived-hMSCs were seeded at 50,000 cells per mL with 4.5 cm^2 per mL of microcarriers in spinner flasks (80 mL volume). To maximize cell seeding, the adhesion step was performed in 50% final volume during the first 24 hours. The percentage of initial cell attachment was similar for both cell types; slightly better cell attachment was observed on Synthemax II compared to CELLstart-coated microcarriers: 48% and 43% for AT hMSCs and 42% to 37% for BM hMSCs. The homogenous cell distribution on the first days of culture resulted in a higher expansion rate with exponential growth from day 4 to day 6 for AT hMSCs and day 4 to day 9 for BM hMSCs. The longer growth phase observed for BM hMSCs resulted in higher cell densities of 350,000 cells per mL compared to 250,000 cells per mL for AT hMSC cultures. Expanded cells maintained their characteristic immunophenotype and multilineage differentiation potential. We anticipate that this xeno-free, ready-to-use microcarrier-based culture technology will enable cost effective, reproducible methods for the scale-up of hMSCs for potential cell therapy applications. References:  Dos Santos F, Andrade PZ, Abecasis MM, et al. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue engineering. Part C, Methods. 17(12), 1201-10 (2011).
B45 Antibody Membrane Switch (AMS) Technology for Facile Cell Line Development Bo Yu, Larix Bioscience, LLC Antibody Membrane Switch (AMS) technology is the most effective and time efficient technology available today for the isolation of production cell lines for therapeutic antibodies or other biological molecules. AMS technology utilizes a unique switch mechanism of alternative splicing and site-specific DNA recombinase to turn cells from expressing membrane-anchored antibodies into production cells secreting the antibody. This enables screening of hundreds of millions of cells per day by FACS. AMS permits selection of highly productive cells with single copy gene integration eliminating the requirement for gene amplification. Utilizing AMS technology can reduce cell line screening time from 8-12 months to 2-3 months. Because AMS generates hundreds to thousands of highly production cell lines, all critical product quality attributes can be assessed at an early stage streamlining downstream process development. The AMS technology has been used to rapidly generate production CHO cells expressing biosimilar antibody Humira in a small scale proof-of-concept experiment. In a 9-week process, ~200 clones were isolated after FACS and then switching off membrane anchorage of the antibody. Four of the top five clones produced more than 300 mg/L of Humira in a 50 ml non-fed shaking culture. The top clone produced 752 mg/L of Humira.
B46 Multiplexed, Structure-driven Biologics Analysis: Robust Methods for all Stages of the Bioprocess Edward Za, Quantum Tessera Consulting The characterization of biologics, biosimilars, and vaccines is critical to regulatory success. It has been made clear that cutting edge science is expected, or required in the case of biosimilars. Reliance solely on chromatographic and spectrometric methods is no longer sufficient. Nuclear Magnetic Resonance (NMR) is the most powerful analytical tool that is applied to biologics. NMR is a structure-based method that is inherently multiplexed, a unique combination of strengths. NMR is an immensely impactful method at all stages of development: from fermentation, process development, to drug substance/product. NMR in many cases is the only way to quantitate these residuals, giving the most robust method to determine what will be CQA/KPAs for many products. The appearance/consumption of media components in bioprocess culture media is a crucial element of understanding growth conditions. NMR can analyze culture media (with minimal sample preparation) in a multiplexed fashion. Routinely, more than 50 media components can be identified and quantitated in a single experiment. This has tremendous impact especially when using poorly defined media components, like hydrolysates. Many biologics need to be folded/refolded. Typically, this process is monitored using indirect methods. NMR directly observes the protein during this proces and, in real time, determines when the protein is fully folded. Using NMR in this way also takes advantage of NMR's inherently multiplexed nature; process residuals are easily detected and quantitated while monitoring the protein. NMR has distinct advantages over spectrometric/chromatographic methods for quantitating heterogeneous residuals, like anti-foam or detergents. For stability and/or formulation studies, NMR again can analyze samples with minimal sample preparation and in a multiplexed fashion. This can be used for release of buffer or investigations of anomolous results. In drug substance characterization, NMR has been shown to be the only technique that gives robust structural characterization of glycans/carbohydrates. In this era of engineering glycan composition/production, the ability to determine the correct structure with direct evidence is incredibly powerful. For biosimilars, vaccines, and some biologics, e.g. insulin, polysaccharides, peptides, etc., the structure of the final drug substance/product has to be determined at the atomic level. NMR is the only structural method that can routinely do this for all of these different product types. We will show examples of NMR used in all these situations.
B47 A Systematic Strategy of Cell Culture Media and Feed Development Meghan McCann, Fujifilm Diosynth Biotechnologies Development of a high-performance and robust fed-batch process for recombinant Chinese Hamster Ovary (CHO) cell lines presents challenges in light of the diverse nutritional requirements observed with different clonally derived cell lines at different production phases. To address these challenges, FUJIFILM Diosynth Biotechnologies (FDB) has developed an innovative strategy of media, feed, and process development methodologies. The efficiency and effectiveness of this strategy have been well demonstrated in multiple Monoclonal Antibody (mAb)-producing CHO cell lines at varied scales, with additional focus on maintaining the balance between product yield and quality. Utilizing a Design of Experiment (DOE) based developmental approach FDB has designed a system to address diverse CHO cell nutritional requirements in a variety of ways. This includes media and feed "tool box" development, media platform development, and media component DOE's that analyze impact of specific components and concentrations on titer and quality. This approach yielded successful cell culture performance at various scales including shake flasks, 2 L bioreactors, and 200 L bioreactors to indicate acceptable process scalability.
B48 Optimization of a Humanized Monoclonal Antibody Productivity using Single Use Bioreactors Tanya Sheinin, Therapure Biopharma Inc. Recombinant antibody and protein-drug conjugates are increasingly being used as therapeutics for the targeted delivery and improved safety of therapeutic drug molecules. Therapure has generated an antibody to human CD163, a cysteine-rich scavenger receptor expressed on peripheral blood monocytes, some tissue macrophages and on a small percentage of CD34+ hematopoietic progenitor cells (Matthews et al, 2006). We have demonstrated that incubation of the antibody (TBI 304) with CD34+ cells and subsequent plating on colony-forming assays allows for the generation of larger, more hemoglobinized erythroid colonies (BFUe) indicating an increase in erythroid proliferation and differentiation. Background: Improving the productivity of CHO cell lines is of critical importance for the manufacturing of therapeutic antibody. Following the successful creation of a stable cell line, an optimized upstream process is required to improve yields of the product of interest. A fed-batch process producing a humanized antibody is presented herein as a case study. The original process in shaker flasks had suitable yields at the end of a 9 day process. However, the same cell growth and productivity was not maintained upon scale-up to a small scale bioreactor. Objectives: Therapure employs well-defined criteria to develop an optimized upstream process to meet titer goals. These parameters include: reactor throughput, specific production rate (SPR), growth rate (seed vs. production), maximum viable cell concentration, and maintenance of high viability. This work focuses on the optimization of culture medium, feeds, feeding schedule, and operational parameters (DO, pH, agitation, etc). Material and Method: The media and feeds are serum-free proprietary formulation purchased from media suppliers. The small-scale reactors (2.5 L working volume) are single use, the bioreactor controllers provide maintenance and control for agitation, temperature, dissolved oxygen, and pH. Upstream samples collected were analyzed for 17 different parameters which included IgG titers, cell density, viability, and metabolites evaluation using the BioProfile Flex analyzer (Nova Biomedical). The parameters which were targeted for optimization were grouped in three and a design of experiment approach was used to identify optimum results. Results: Preliminary results show an increase of 2-3-fold over the original process.
B49 Implementing a Novel OPC Connectivity Solution for Improving Bioprocessing Monitoring and Control William Mil, Flownamics OPC communication provides process automation connectivity and interoperability between various analytical instrumentation and supervisory control and data acquisition (SCADA) systems. A novel OPC communication device, FlowWeb™ OPC Controller, was developed to improve and/or increase functionality of existing bioprocess SCADA applications. This device was designed to interface multiple analytical devices, regardless of communication interface, to any OPC-enabled SCADA and facilitate a more comprehensive information management system for bioprocess monitoring and control. Real-time data from an on-line biochemistry analyzer, in-line turbidity sensor, in-line gas analyzer and at-line analytical balance were simultaneously collected and integrated into a Sartorius BioPAT® MFCS SCADA application. Each instrument required either a serial, TCP/IP or analog signal to send its respective data to the OPC Controller, which processed the streaming data for OPC communication. OPC-tagged data were subsequently sent to the MFCS SCADA via the FlowWeb OPC Server/SCADA OPC Client interface. The FlowWeb™ OPC Controller technology optimized bioprocess knowledge, monitoring and control by: 1) providing real-time data acquisition of in-line, on-line and at-line analytical instrumentation; 2) improving data management through integration and centralization of critical process information; and 3) allowing remote control of analytical instrumentation through the host SCADA application.
B50 A single, chemically-defined culture medium supporting a full-range of development activities for the production of biologics Mark Arjona, Irvine Scientific BalanCD™ Growth A was designed as a high-performance CHO production medium suitable for batch and fed-batch processes. Its compatibility and superior performance in subcloning applications allows for the use of a single medium during multiple stages of development, assuring a seamless transition from one stage of development to the next. With the multitude of commercially available production media and relative scarcity of subcloning media, BalanCD™ Growth A represents a unique, chemically-defined solution for the production of biologics.
Formulation and Delivery
C1 Bispecifics: Insight into a Complex Degradation Profile Leanne Amery, MedImmune mAb1 is a bispecific antibody exhibiting loss of activity under thermal stress conditions. A putative link was made between ~30% photo-oxidation of a specific Trp in the light chain CDRs and a ~60% loss in activity. Data is presented to test the hypothesis that Trp oxidation during thermal stress caused the observed loss in potency. A 12 week stability study was performed with and without Met antioxidant in the formulation buffer. Samples were tested for potency and analysed by HP-SEC to monitor aggregation. Reduced peptide mapping was used to determine oxidation levels at a number of Trp and Met residues and deamidation at specific Asn residues. The results of this study revealed addition of Met as an excipient reduces oxidation, but does not prevent loss of activity. Surprisingly, in samples without Met, up to 50% loss of potency was observed despite Trp oxidation remaining relatively low. Increased levels of a deamidation intermediate at an Asn site were observed under thermal stress. These observations weaken the putative link between Trp oxidation and loss of potency under thermal stress conditions, instead indicating that a distinct chemical degradation pathway (deamidation/stable succinimide formation) reduces bioactivity.
C2 The use of temperature dependent intrinsic fluorescence for the analysis of in-process stability of a protein Walter Ausserer, Pall Life Sciences The manufacturability of proteins is heavily influenced by their stability to the processing conditions required to purify and formulate them. It would therefore be very useful to have a fast, efficient method for screening a wide range of manufacturing conditions to discover the optimal conditions for each step and, more importantly, the conditions to avoid. To demonstrate this approach a study was undertaken using in-process samples of a monoclonal antibody from a conventional purification development. Each stage of the process was assessed using a DOE approach to map the design space and to find the optimal processing conditions. In parallel, a stability assessment of the antibody at each stage was carried out using the Optim® instrument to determine the overall manufacturability of the product as impacted by the Concentration of the protein in solution, the buffer in which it is stored (pH and/or conductivity as appropriate) and the stage of the process.
C3 Orthogonal Approaches to Higher Order Structure Elucidation During Formulation Matrix Design Fiona Greer, SGS Life Science Services The use of higher order structure biophysical techniques within biopharmaceutical formulation development is rapidly gaining greater significance compared to conventional biochemical techniques. Formulation scientists realise that maintaining structural integrity of the biopharmaceutical drug product and reducing molecular aggregation is key to maintaining biological function, hence increasing product shelf life. As biological molecules are more complex than small drug entities, more sophisticated techniques are required for their characterisation. We demonstrate the application of orthogonal biophysical analyses (Near-UV CD, DSC, SV-AUC, SEC-MALS, FT-IR and DLS) in formulation design using two model proteins, Bovine Serum Albumin (BSA) and an IgG. BSA is known to form oligomers in solution under ambient conditions, and was chosen as the model for size estimation studies. We demonstrate a full particle sizing study of the protein using SEC-MALS, SV-AUC and DLS. IgGs are the most common API of biopharmaceutical products in development and on the market and due to their complex multi-domain nature these proteins make a useful model for assessment of changes in higher order structure when stored in a variety of formulation matrices. We demonstrate an evaluation of conformational stability of an IgG in two common buffer systems using near-UV CD, DSC and FT-IR.
C4 Formulation Strategies for Human Serum Albumin Fusion Protein Therapeutics Yen-Huei Lin, Teva Biopharmaceuticals USA Human serum albumin (HSA) fusion proteins present a major class of fusion protein therapeutics with characteristics of enhancing serum half life of its fusion partners of diverse size and functions. However, the diverse nature of HSA fusion partners also makes it a challenge to develop stable and robust products for fast-track into clinical studies for HSA-fusion therapeutics. To meet the formulation challenge and accelerated entry into clinical trials, a platform approach was applied to 5 different HSA fusion molecules, including peptide, cytokine, and therapeutic enzyme as fusion partners. This early stage platform formulation strategy applied a sugar/polyol based formulation, conservative lyophilization cycle with a pre-selected platform container closure system. Drug products at 2-100mg/mL developed with this platform were stable for at least 24 months at 2-8 °C. In addition, the platform formulation was proven feasible for optimization into high concentration liquid commercial product stable for 36 months in pre-filled syringe. This poster presents these formulation strategies that enable accelerated entry for clinical development and product specific commercial optimization for HSA fusion protein therapeutics.
C5 Development of a small-scale freeze/thaw model to emulate manufacturing-scale freeze/thaw conditions of bulk drug substance storage bags Sumati Mattoo, Biogen Idec Flexible, single-use bags (such as the Celsius®-Pak line from Sartorius Stedim) have become a popular choice for storage of biopharmaceutical bulk drug substance. Freeze/thaw times in the manufacturing-scale bags (i.e. 2L, 6L, 12L) can be long, which may induce cryoconcentration and stability issues for the API. Evaluation of freeze/thaw stability at manufacturing-scale is often hindered by lack of bulk drug substance availability in such large quantities. A small-scale freeze/thaw model that emulates large-scale freeze/thaw times was developed using a bench-top controlled freeze/thaw system and 30mL bags.
C6 A Process Description Tool (PDT) for a Formulation Development Annette Heydenreich, Novo Nordisk A/S The formulation development department at Novo Nordisk is a high throughput project driven organisation and responsible for the formulation development and documentation from first human dose to end of phase III, including all supporting CMC activities. Thus, a tool for align processes and share knowledge across projects is required. In this poster, a process description tool (PDT) will be presented, an intuitive and lean way to share best practices, recommendations and information on all department-related processes. The tool includes following technical features • Searchable PDF-file for electronic use • Navigation through hyperlinks • Aligned with project manual • Visual table of content, based on a generic project timeline • Each process described via the same template • Living tool - Build-in system for updating the file Conclusion • PDT is the tool that describes, connects and anchors processes and activities in the department - both project-specific and not-project-specific processes • PDT is for guidance only, but refers to SOPs and regulatory requirements where appropriate • Is continually updated, by everybody in the department
D1 Development and Implementation of a Process Control Strategy for a Legacy Biologics Product Daniel Carraher, Genzyme A robust Process Control Strategy (PCS) as touched upon in several regulatory guidance documents, including ICH -Q8R2, Q10, Q11 and FDA's Guidance for Industry Process Validation: General Principles and Practices, is becoming an increasingly integral component of the biopharmaceutical manufacturing industry. There are no specific industry guidelines prescribing PCS terminology and approach. These can vary within the biopharmaceutical industry according to the point in the process/product lifecycle in which the PCS is being developed, and thus the extent of development and manufacturing history available. Genzyme has been incorporating PCS and Quality by Design concepts into the development strategy for new products. An effort has recently begun to bring Genzyme's legacy biologics products into this framework. In this presentation, we will provide a strategy for PCS development and implementation for biologics products, which is applicable to products with dedicated production facilities and to those products manufactured at multiple sites and scales. This presentation will include the approaches taken to address particular challenges encountered when implementing a PCS for a legacy product with an extensive history of commercial manufacture within Genzyme's Framingham Biologics organization.
D2 Platform Purification of a Domain Antibody Anna Heijbel, Ge Healthcare The production and purification of monoclonal antibodies (MAbs) is nowadays a mature industrial process. Commonly, platform approaches are used with Protein A affinity chromatography as the capture step. Now, other molecules are also emerging as possible alternatives to MAbs, namely antibody fragments (e.g. Fab, scFv, domain antibody [dAB], etc.). Most of these molecules lack the Fc part of the antibody making a platform purification approach using Protein A impossible. However, with the introduction of novel affinity medium new possibilities have emerged. In this poster, a three-step purification process for the purification of a dAB is presented. The process is based on the recently introduced platform for the purification of kappa light chain antibody fragments. The purification process was developed with QbD in mind, therefore including high throughput development (HTPD) tools and DoE to achieve an understanding of the effects of different factors in the experimental space.
D3 Risk Mitigation Through Component Material Selection Including Extractable Data from Materials of Construction Mike Johnson, Entegris With the continued growth of single-use bioprocessing systems comes increasing concerns associated with today's polymers. Through a more fundamental understanding and evaluation of polymer materials, it is possible to signficantly reduce the potential for final product contamination. This poster presentation will discuss and compare key polymer material performance characteristics as well as present extractable data from different classes of polymer materials used as wetted surfaces in single-use systems.
D4 Scale-up study of mAb manufacturing process from 200 L to 2000 L bioreactor Hideyuki Kajihara, Takeda pharmaceutical company limited There are many process parameters in animal cell culture and they are classified into two types, scale-dependent or independent parameters. Among them, agitation speed and aeration rate are very important scale-dependent parameters, so they should be prospectively determined for large-scale culture. Based on our previous works using the 200 L bioreactor, process parameters for the 2000 L bioreactor culture were predetermined through calculation method. We applied them to 2000 L scale culture and checked its validity and manufacturing capability. As a result, production culture was carried out as expected. The growth profile was comparable between 200 L and 2000 L scale. The mab titer obtained at harvest, 6.07 g/L, was also comparable to the titer at the smaller scale bioreactors.
D5 Lessons Learned Experienced during Back to Back (to Back) Technical Transfers to an International Biotechnology Manufacturing Facility Vincent Lau, Biogen Idec Leading a Technical Transfer for an early clinical phase program into a newly operational International Biotechnology Manufacturing Facility encompasses multiple challenges. While execution of a single program to an International Facility is challenging, implementation of multiple sequential programs with overlapping Technical Transfer activities requires a monumental effort highlighting the need for a cohesive team built upon strong relationships to ensure success at the manufacturing scale. An overview of lessons learned detailing specific hurdles experienced with overlapping Technical Transfer activities will be presented.
D6 Characterization of kLa Using Definitive Screening Designs for Fermentation Scale-Up Weng Long Lin, Lonza Biologics Inc Fermentation development and optimization are typically performed at small scale to reduce their cost and time requirement. High cell density aerobic fermentation is scaled-up often based on keeping volumetric mass transfer coefficient (kLa) constant. To facilitate a successful transfer of an optimized small-scale fermentation process to lab, pilot, and industrial scale, the kLa profiles for these fermentors should be determined. Although a number of correlations and predictive models for kLa are available, they do not have universal applicability. The purpose of this study was to use a statistical Design of Experiments (DoE) approach to characterize kLa in 1.3L DASGIP, 10L Biostat B, 40L Biostat CDCU and 200L ABEC microbial fermentors, and hence to use this information for fermentation scale-up. Polarographic dissolved oxygen sensors were used to measure dissolved oxygen concentrations in the fermentors. kLa was determined in de-ionized water by using the dynamic re-oxygenation method. Definitive Screening Designs, a new efficient statistical Design of Experiment, were used to characterize kLa in the fermentors. Four experimental factors, temperature, agitation, air flow rate, and volume, were included in the experimental design as input parameters, and kLa was the output parameter. Definitive Screening Designs showed that agitation, air rate and volume were statistically significant for all the fermentors. Air and volume had strong positive correlations with kLa, and agitation had strong quadratic effects on kLa. Results showed that four fermentors have comparable kLa profiles. The correlations determined here will be used as an effective tool for fermentation development, scale up and tech transfer.
D7 Use of a Simple Visual Tool to Monitor Commercial-Scale Purification Process Performance on the Manufacturing Floor Lakshmi (Prasad) Pathange, Bayer HealthCare Tools such as standard work and daily weekly operational reports are used in the manufacturing to manage floor operations. These tools play an important role in facilitating communication between floor operators and management. They are used by operators to track, monitor and improve floor operations. Though these tools are excellent in analyzing operational efficiency, they rarely contain details to monitor purification process performance. In order to enhance floor operator involvement in process performance issues, a simple visual tool was developed. Chromatographic data such as UV peak height, conductivity and pressure were used to generate a process monitoring tool. The data entry and compilation are performed by operators on the floor after each process step. The data is continuously reviewed by operators and management to identify any emerging issues that could impact process performance. Case studies will be presented to demonstrate its use by operators and management as simple and effective tool to communicate and evaluate commercial-scale purification process performance.
D8 Evaluating the Impact of Controlled Nucleation on Lyophilized Product Attributes and Processing Characteristics in an Aseptic Environment Wendy Sunderland, Lyophilization Technology, Inc ControLyo™ Nucleation on Demand technology (ControLyo) is an innovative technology that improves the uniformity of nucleation of ice in product vials during freezing within the lyophilizer. Processing of model crystalline and amorphous systems was performed in a 48-square foot lyophilizer in an aseptic environment. Lyophilization cycles were performed for both model systems using either ControLyo or a standard freezing method, resulting in product vials nucleating simultaneously and randomly, respectively. The product vials from both studies were then dried using identical primary and secondary drying temperatures, pressures, and times. There is significant work in the literature of laboratory studies evaluating controlled nucleation of ice on resulting product characteristics; however, there is limited, if any, research including product processed in an aseptic Good Manufacturing Practices (GMP) facility. The studies encompassed product temperature data and finished product characteristics of two model systems processed in an aseptic facility. In particular, effects of controlled nucleation and freezing, sublimation, and desorption will be compared. These will be reflected through product temperature data and physical appearance, reconstitution behavior, pH, and residual moisture content. Early indications are there are subtle differences in some lyophilized product attributes between samples utilizing ControLyo and samples utilizing a standard freezing method. The data presented will offer insight into the effect on freezing and drying behavior, as well as finish product characteristics of controlled ice nucleation technology compared to the standard freezing method.
Recovery & Purification
E1 Small Scale Protein Purification for Rapid Optimization of Biotherapeutic Protein Production Andrew Barry, PerkinElmer Optimization of expression and purification conditions presents a key challenge towards the development of processes to efficiently produce biotherapeutic proteins. The ability to screen matrices of conditions critical to protein identity, titer, structure, and function requires systems for rapid purification and analysis. Small scale protein purification enables increased sample throughput and reduced purification time, resulting in faster, more efficient optimization for biotherapeutic process development. These advances enable the use and scaling of novel resins through functional dynamic inding capacity testing for ion exchange and affinity chromatography. Here, we will discuss automated purification using three different modalities, each with benefits of loading mass, speed of purification, and flexibility of resin choices and sample throughput.
E2 Residual DNA Testing - design, Validation, Optimisation and Overcoming Sample Matrix Interference Paul Byrne, Covance Labs The assessment of residual DNA during the manufacture and purification of biologics from mammalian cells is important to ensure that final products/drug substances (test material) contain acceptable levels of residual DNA. Developing and validating Quantitative Polymerase Chain Reaction (QPCR) assays for detecting residual DNA can be challenging and time consuming. Our Biotechnology group has developed significant expertise in designing, validating and optimising residual DNA (RDA) assays to achieve the required sensitivity and overcoming sample matrix interference. The current requirements for developing and validating RDA assays will be discussed, including various solutions to overcome sample matrix interference.
E3 Next Generation Polishing Technologies for Robust Host Cell Protein and MAb Aggregate Removal William Cataldo, EMD Millipore Flow-through polishing technologies can enable a faster, simpler, and more economical purification of monoclonal antibodies. We will discuss how a set of next generation polishing technologies can allow for robust removal of product and process-related impurities, and for elimination of a second bind/elute chromatography step. Novel adsorptive technologies operate by the mechanisms that are complementary to traditionally deployed anion-exchange media, thus enabling easy removal of product aggregates and HCP populations that traditionally can only be removed using bind/elute chromatography. Experimental results with multiple MAb's demonstrate robust performance, including maintaining high product yield, removal of HCP, and aggregates. The new approach could enable a simplified and more powerful platform for MAb polishing than existing technologies.
E4 Efficient Separation of Antibody Light Chains From Bi-specific Antibody Monomer Using Mixed-mode Sorbents Yamuna Dasarathy, 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
E5 CaptureSelect® Affinity Chromatography Ligands and Resins for Laboratory and Process Scale Applications Bruce Dawson, Life Technologies Corporation CaptureSelect® affinity ligand and chromatographic resin products provide novel & cost-effective solutions for affinity purification challenges. CaptureSelect® products improve existing purification processes by increasing yield, reducing process steps, eliminating trace contaminants, and replacing inefficient capture steps limited by small molecule, bulk property, and immunoaffinity ligand technologies. Using single domain heavy chain antibody fragments, CaptureSelect® technology combines the intellectual property and expert knowledge to discover, develop, and optimize affinity ligands and resin for specific needs, as well as standard products for common biotherapeutics such as antibodies, antibody fragments, vaccines, and biosimilars.
E6 Accelerating MAb Downstream Process Development Using a Pre-Selected PD Kit Chase Duclos-Orsello, EMD Millipore Corporation With the proliferation of monoclonal antibodies in preclinical and Phase I stages, speed to clinic continues to be an essential component of determining commercial success. Many biotherapeutic manufacturers use a templated set of purification unit operations in order to streamline process development activities. In many cases, however, even further time savings are possible in process development by fixing not only the unit operations but also certain process parameters. In this case study, the purification process for a monoclonal antibody is developed with the specific intent of minimizing effort in order to speed the time to producing clinical material. The process development strategy is discussed in detail as well as the subsequent use of off-the-shelf single-use systems and assemblies for manufacture of clinical phase material.
E7 A Case Study: 3-Step Process for Efficient Mab Purification Using Different Commercially Available Chromatography Resins Melissa Holstein, EMD Millipore This study showcases a portfolio of commercially available biopharmaceutical chromatography resins designed for the efficient purification of monoclonal antibodies. A three-step purification process has been implemented which showed effective removal of the main contaminants, low ligand leakage, and high yields over the entire process. Two Protein A affinity chromatography resins were evaluated as the first step in the process. One of the resins is based on controlled pore glass while the other is a rigid polymeric resin. The Protein A elution pools were further purified using cation exchange chromatography. Two cation exchange resins with different selectivities were compared. The final purification step consisted of either an anion exchange chromatography resin or an anion exchange membrane adsorber. The product yield, HCP removal, leached Protein A removal, and aggregate content were evaluated at each step in the process. Final process yields ranged from 80 to 90 percent. This work highlights the purification capabilities of a collection of resins designed for efficient Mab purification.
E8 Versatile Protein Coating on Magnetic Particles for Bioseparation Charlie Huang, Anteo Diagnostics Ltd Recent advances of using magnetic particles for bioseparation create a need for better activity and stability at the surface interface between the synthetic particles and capturing agents. However, direct chemical coupling (e.g. EDC/NHS chemistry) of proteins to superparamagnetic particles can cause damage, and the process is often lengthy and difficult to control. We developed Mix&Go™, a novel chelation-based surface chemistry that enables simple, fast, and gentle protein binding on virtually all particles used in bioprocessing. Here the effects of a one-step, one-hour Mix&G™ activation of superparamagnetic particles, and their successful coating or co-coating of various proteins, e.g. Protein A/G, antibodies and streptavidin for bioseparation applications are discussed
E9 Characterization of an Experimental High Capacity and Alkaline Stable Recombinant Protein A Resin Chinlun Huang, Tosoh Bioscience LLC Therapeutic monoclonal antibodies (mAbs) have become a major product class in the biopharmaceutical industry, and they are expected to be a source of new therapeutics. In addition, mAb expression levels are increasing, and this increase is leading to increased demand for resins with higher binding capacities. This experimental high capacity recombinant protein A resin was developed by Tosoh Corporation in response to the demand. The experimental resin utilizes a recombinant, alkaline stable Protein A variant obtained from a third party vendor. This new non-animal derived ligand is bound to the base bead using a multipoint attachment. The mean particle size is 45 µm, and the average pore size is 100 nm. This resin has a pressure rating of 0.3 MPa, and it is stable in the pH range 2-13 for short periods of time. This experimental resin achieved a static binding capacity of 80 g/L for human IgG and a dynamic binding capacity of approximately 70 g/L. In this poster, properties of the experimental resin, including pressure characteristics, purification of monoclonal antibody from cell culture supernatants, and a CIP study with sodium hydroxide will be presented.
E10 A Scalable Method for Packing Chromatography Columns Ganesh Iyer, EMD Millipore Currently the bio-pharmaceutical industry is heading towards cost cutting, time savings and single use manufacturing approach. This has opened up a whole new market for disposable technologies. Pre-packed disposable chromatography columns are a best fit for preclinical and clinical stage Monoclonal Antibody (mAb) production and vaccine manufacturing processes which require quick turnaround times and aggressive validation respectively. This work describes development of pre-packed and ready to use chromatography columns to meet this need in the industry. A robust packing method was developed to pack two different types of resins of different particle sizes, one with a polyacrylate base matrix and the other with a polyvinyl ether base matrix. The column performances were evaluated by measuring height equivalent theoretical plates (HETP), asymmetry, pressure-flow characteristics and protein retention. The results showed that the method is reproducible and scalable over a column volume range of 1.6 to 16 lit. Further, the columns retained their performance characteristics even after sever vibration and drop tests. This suggests that the method developed and the column designs are desirable for a pre-packed and disposable chromatography format.
E11 Evaluation of Feed Flow Geometry in Hydrosart® Cassettes with Protein Solutions Hamanth KALIGOTLA, Sartorius Stedim Biotech, North America Sartorius Sartocon® cassettes are available with the option of two internal flow geometries. Sartocon cassettes with "E" type spacers are designed for high protein concentration applications and for viscous products; while the cassettes with "ECO" screens are designed for protein concentrations less than 15% (150g/L) and for other products with low viscosity [<3cp (3mPas)]. With current mAb harvest targets of ≥5 g/L and increased use of subcutaneous dosages, protein concentrations often approach 200 g/L or more. It is therefore important to understand when to use each type of cassette and the implications. This evaluation demonstrates that "ECO" channel cassettes can achieve twice the flux at 1/3rd the feed flow rate of the "E" channel cassette. The "ECO" cassettes feature 26% more surface area per standard cassette width; making it possible to install more area in the cassette holder.
E12 What is the Most Productive Protein A Media for Therapeutic Antibody Manufacturing? Takuo Kawase, Chugai pharmaceutical Co., Ltd Protein A based affinity chromatography is the industry standard for therapeutic antibody purification and is the biggest cost driver in raw materials of downstream process. MabSelect SuRe™ (GE Healthcare) is the most commonly used Protein A media, however, various types of products have been launched and now biopharmaceutical manufacturers have more choices to increase productivity of the Mab manufacturing process. Productivity of the Protein A chromatography process is defined by equally important four factors; capacity, flow rate, reusability and price. We evaluated 16 brands of Protein A media and calculated predefined productivity index based on supplier's technical information. Then actual productivity and quality performance were experimentally investigated using the scale down model. Our experiment results showed one of the Protein A media tested has 2 to 3 times higher productivity than MabSelect SuRe™. Since Non-Protein A purification methods have been still challenging to integrate into Mab platform process, to employ newly introduced Protein A media is an effective way to decrease production cost of antibody manufacturing without any significant changes of process and facility.
E13 New Strategies for High Throughput Label-free Characterization of Biotherapeutics Yixin Lin, Pall ForteBio To address the needs of a large number of biotherapeutic molecules and vaccines in development, it has become increasingly important for biopharmaceutical companies to adopt a new generation of high throughput (HT) analytical techniques to facilitate fast and accurate characterization and processing monitoring. Here we present new label-free strategies not only to characterize the functions of biomolecules, but also their stability and aggregation status, all in HT format. Recent advances in the use of Bio-layer Interferometry (BLI) are described for monitoring functional roles of molecules through quantitation and kinetic characterization, with emphasis on new development/methods in bioprocessing. In addition, we describe a micro-volume HT platform to monitor and characterize protein structural stability and aggregation using intrinsic florescence and static light scattering, in one single experiment. This approach has been used to optimize in-process conditions during purification and formulation. The combination of these strategies provides comprehensive analyses of biomolecules at various stages of drug development, with much improved speed, ease and reliability, and lower cost.
E14 Implementing Disposable Chromatography: Technology Fit in Downstream Purification Fletcher Malcom, Repligen Many of the world's leading biopharmaceutical companies have adopted single use and disposable technologies for faster product changeover, favorable economics, and improved safety. While single-use and disposable technologies are prevalent in many areas within upstream and downstream processing, up until now there has not been a broadly applicable solution for chromatography steps. In this poster, overcoming the four most critical barriers to implementing pre-packed disposable columns in clinical manufacturing is discussed. • Column Size: A disposable chromatography solution for downstream purification of biomolecules is introduced. The importance of column size is evaluated. • Chromatographic Performance: Although pre-packed columns are designed primarily as campaign-use/disposable technology, biological and chemical compatibility of the hardware makes them suitable for multiple uses. An example of a cycling study for purification of a biomolecule is presented. •Economics: An economic model developed with BioProcess Technology Consultants (BPTC) justifying the cost benefit of pre-packed columns is presented • Documentation: The documentation components required to support an NDA and CMC package are noted.
E15 Effective mAb Purification Process without Buffer Exchange Step Combining Packed-Bed Chromatography and Anion-Exchange Membrane Adsorber Tomokiyo Marumoto, Asahikasei Medical Co.,LTD Purification of monoclonal antibody (mAb) is quite important for both production and research point of view. Packed bed column chromatography process including protein A column is commonly used for mAb purification in which buffer exchange steps by UF/DF are possibly desirable between the individual column steps. Although the buffer exchange step is important for the higher performance of column process, it increases buffer consumption and process time. The combination use of high binding capacity cation exchange (CEX) media in bind-and-elute mode and salt tolerance type of anion exchange (AEX) media in high speed flow-through mode is thought to be a positive measure for these issues. In this report, we evaluated mAb purification using new type of macro porous cellulose chromatography media and hollow fiber type AEX membrane adsorber. The elution pool from protein A column was diluted with deionized water and applied to the next chromatography steps. Cellulose CEX resin shows quite high mAb binding capacity (10% DBC >100 mg/mL-media) even for the conductivity of 5 to 8 mS/cm. In the flow-through purification, hollow fiber membrane adsorber showed most effective reduction of HCP and leached protein A compared with other chromatography resins.
E16 Rapid and Highly Efficient Downstream Purification of a Recombinant Humanized Monoclonal Antibody from CHO Cells Mukesh Mayani, Therapure BioPharma A downstream purification process that yields a high quality humanized monoclonal antibody (HuMAb) is a major challenge that has significant implications for the production of material suitable for use in humans. Presented here are key aspects for the efficient production and purification of a recombinant HuMAb produced by CHO cells in culture. A titre of approximately 2 g/L recombinant HuMAb was obtained with minimal parameter optimization during upstream development. Downstream process optimization resulted in a rapid and highly efficient purification process. The purified HuMAb product at at the 5 L scale contained very low levels of process- and product-related impurities, for example, 0.9 ppm host cell proteins (typical limit: NMT 100ppm), <0.5 ng of host cell DNA/dose of HuMAb (typical limit: NMT 10ng/dose) and 0.4 ppm of leached protein A (limit: NMT 10ppm). The endotoxin value in the purified HuMAb was found to be <0.2 EU/mg of HuMAb (Limit: 0.24 EU/mg) with 98.8% purity by SEC-HPLC method. The aggregate level as determined by SEC-HPLC was less than 1.2%. Currently, the highly efficient purification process has been scaled up at a 50 L pilot scale using single-use disposable technology to generate approximately 70-80 grams of HumAb at an expected 80% yield.
E18 Introduction and Evaluation of a Novel Alkali Stable Protein A resin - Amsphere Protein A JWT203. Masayoshi Nagaya, JSR Life Sciences Amsphere Protein A JWT203 is a novel protein A media designed for the capture purification step of monoclonal antibody manufacturing by JSR Life Sciences. It is a combination of JSR Poronomics, our proprietary technology for the design of hydrophilic and porous resins with large surface area, and a novel recombinant Protein A with high alkali resistance. JWT203 can capture various antibodies and Fc-fusion proteins with high dynamic binding capacity, comparable to the leading commercially available protein A media. High purification efficiency is observed with this resin, leading to low DNA and host cell protein content in the elution product pool. ELISA analysis suggests low protein A leachate levels from the Amsphere product as well. JWT203 product has been tested at both 0.5M and 0.1M NaOH enabling significant reuse of the resin at commercial scales. JWT203 matrix is about five times harder than agarose-based matrix when compared using a compressive elastic modulus measurement. The hard base matrix of JWT203 makes column packing easy and hydrodynamic properties ideal for flow packing at any scale. The strong technical and business case of Amsphere Protein A JWT203 makes this resin an ideal candidate for transparent and seamless scale up from early phase clinicals to use in commercial manufacturing processes of therapeutic antibody products. In this poster, a full study on fundamental performance of JWT203, including DBC vs flow rate, intermediate washing conditions and CIP with aqueous sodium hydroxyl solution is explained. With any new resin, packing is a challenge due to the difference in handling and methodology. This study further addresses the packing studies conducted at JSR laboratory comparing lab and process scale column packing. In addition, a case study using a pilot scale column is explained to show packing proof of concept at larger scale.
E19 Implementation of a Depth Filter for HCP Clearance in a Recombinant Protein Purification Process Saravanamoorthy Rajendran, Alexion Pharmaceuticals Implementation of a Depth Filter for HCP Clearance in a Recombinant Protein Purification Process: Saravanamoorthy Rajendran, Trung Thieu, Jessica Statkus, Jeffrey Zugates, Rachael Alford Purification Development, Alexion Pharmaceuticals Charged depth filters have historically been used in cell culture clarification for removal of cell debris. They have also been used for turbidity reduction after Protein A step in monoclonal antibody (mAb) purification processes. The work presented here demonstrates the capability of a charged depth filter to reduce host cell proteins (HCP) in a recombinant protein purification process with a high level of HCP. Multiple charged depth filters were screened for their efficiency to reduce the HCP levels using scaled-down models. Impact of depth filter type and filter throughput on HCP clearance were identified. The depth filter that provided the highest HCP clearance was implemented into the purification process. Subsequently, the placement of the filtration step in the purification process was evaluated. The results showed that the location of the depth filter in the purification process was important to meet the target HCP levels. The resulting purification process was successfully scaled up to the 50 L production scale with HCP levels less than 100 ng/mg.
E20 Disruptive Technologies for High-performance Downstream Processing Fabien ROUSSET, Novasep Disruptive approaches for the purification of biologics are necessary to remain competitive; it has become essential to increase process performance while remaining cost-effective. This presentation will cover the main unit operations within a downstream line and highlight ways to increase performance of the whole process. An economic and productivity comparison will also be made between a traditional process design and a process with disruptive technologies. Whether focusing on capture with protein-A resin or implementation of continuous chromatography and single-use TFF, great improvements can be made at every step.
E21 Merrimack Pharmaceutical Evaluating Acoustic Wave Separation for Mab Harvest CHO Cell Clarification John Rozembersky, FloDesign Sonics Mammalian cell densities and product titers in bioreactors continue to increase due to the notable advancements made to the upstream expression systems for recombinant therapeutic proteins, monoclonal antibodies, and vaccines. These achievements are creating unprecedented challenges to the clarification and downstream processes (DSP). Considering the complex and inconsistent feed properties of the cell culture delivered from the bioreactor including the large amounts of biomass and increased levels of contaminants and cell debris, developing a "robust" process using current technologies (DFF, centrifuge, TFF) is and will continue to be a challenge. Hence, the need to look to new separation technologies for cell clarification that exceeds current performance expectations while remaining economically viable is a must. The technology that Merrimack is evaluating for the primary clarification process prior to sterile filtration is acoustic wave separation (AWS). The technology used for the investigation is the Wave D3TM separation technology developed and patented by FloDesign Sonics Inc. (Wilbraham, MA). In this article, results will be shown comparing the performance of a non-optimized scaled-down AWS process to that of the full-scale DFF GMP process. Cell clarification efficiency and product quality were the focal points in this trial study.
E22 Innovative Pre-Packed Process Columns for Biomanufacturing Tim Schroeder, Atoll GmbH Greater adoption of single-use and disposable systems in biomanufacturing and development is going to require improved downstream device innovation in particular for pre-packed chromatography columns. We developed and evaluated a disposable, pre-packed and pre-qualified column platform MediaScout MaxiChromAC suited for the purification of biopharmaceutical compounds such as mAbs and vaccines in preclinical, clinical phase I and II studies as well as full scale manufacturing depending on scale and titer of the fermentation process. The innovative design of the column allows direct connection to state-of-the-art disposable chromatography systems as well as the packing of a broad range of commercial available chromatography media of different particle sizes including rigid, semi-rigid and soft polymeric backbones. Transparent column housing allows top to bottom bed inspection during the chromatographic workflow. This outline is showing a complete packing evaluation for a 14.1 L pre-packed MaxiChromAC column including HETP and As measurements prior and after ground and aircraft transportation
E23 Progress on a Fully Disposable Downstream Platform: A Simple, Plug-in Solution to the Downstream Bottle Neck for Flexible Facilities and Traditional Manufacturing of mAbs. James Stout, Natrix Separations, Inc. Natrix Separations, Inc. is developing functional hydrogel polymers and pre-packaged single use cartridges to meet the chromatography challenges of today and the future. Natrix's patented Advective™ flow technology features polymeric hydrogel formed within a flexible porous support matrix. The support matrix provides mechanical strength, while the hydrogel properties determine the separation chemistry of the product. An advantage of the Natrix chemistry is the ability to place virtually any functional group chemistry throughout the hydrogel polymer. Therefore, the Natrix technology combines the superior binding capacity of conventional resin-based columns with the high throughput of membranes in a single-use format that eliminates costly packing, cleaning, validating and other burdens of conventional methods. This talk will focus on the progress to date on building a fully disposable process that competes well against a ProA process for mAbs. There will be two different device formats and chemistries discussed. The first is a positive binding format using weak cation exchange chemistry to capture mAb from clarified cell culture. The second format is negative binding flow through strong anion exchange as a final polish for finishing removal/reduction of impurities in the mAb process prior to formulation as drug substance. Lastly, some comparison data and calculations showing process efficiency and differences between a ProA process and a Natrix non-ProA process will be discussed.
E24 Overcoming Artifacts During Virus Filter Validation: A Case Study Arleene Velayo, Gilead Sciences Millipore's Viresolve Pro viral filter (VF) is often incorporated in antibody purification platforms for its ability to achieve high filter throughput and stable flux while demonstrating robust viral reduction properties. Small scale process validation studies are performed not only to demonstrate its capacity for viral clearance but also to determine achievable volumetric throughput. Achieving target throughput is crucial as it dictates load density parameters and the exorbitant cost of commercial manufacturing virus filtration. In this case study, the viral filtration of product intermediate during process validation, resulted in poor performance that failed to achieve target throughput. Particle formation was observed in feed material as a result of normal handling conditions typical of traditional small scale viral clearance studies (adsorptive prefilter decoupling, post spike sterile filtration, pouring, etc). The formation of particles, some greater than 10 microns (detected by FlowCam and DLS), is believed to have caused filter fowling and decreased VF performance. Only with special, unconventional, practices was it possible to mitigate particle formation. Decreasing the air liquid interface by utilizing a pump for liquid transfers, extreme gentle handling, and holding intermediate feeds for a duration of time, helped to achieve the target volumetric throughput for a successful process validation.
E25 Development of AlphaLISA assays for Quantification and Characterization of Biotherapeutic Proteins and Process Contaminants Greg Warner, PerkinElmer Inc Immunoassays are commonly used in the development and characterization of therapeutic proteins and monoclonal antibodies (TAbs). There is an increasing need for efficient and sensitive assays to detect and characterize these proteins and TAbs during various stages of development. The most common technology for performing immunoassays is the enzyme-linked immunosorbent assay (ELISA), which is a robust method but requires multiple and time-consuming steps that can lead to variable data. The AlphaLISA® assay format is a chemiluminescent homogeneous bead-based technology which does not require washing or separation steps, and as such provides significant advantages over ELISA. AlphaLISA assays are normally performed in 96- or 384-well plates and are run with sample volumes typically around 5 microliter, conserving valuable test materials. The total assay time is less than 3 hours. Several new AlphaLISA assays have been developed to quantify and characterize biotherapeutic proteins and Tabs purity including: • Detection of bioprocess contaminants such as host cell proteins (CHO and PER.C6® HCP). • Quantitation of Residual Protein A from chromatography purification of TAbs. Data provided here demonstrate that AlphaLISA technology provides an alternative and versatile assay platform to ELISA and can improve the workflow of various analytical assays in the modern biotherapeutics development laboratory.
E26 Development of a Flow-through Mode Cation Exchange Process for the Purification of a Monoclonal Antibody Rachel Wollacott, MassBiologics Cation exchange (CEX) chromatography is commonly used as a polishing step in the purification of monoclonal antibodies (MAb) due to its capacity to bind product-related impurities, such as MAb dimers, and process-related impurities, such as host cell proteins and DNA. This process is typically run in bind-and-elute mode with dynamic binding capacities on the order of 50-100 mg/mL resin. During process development activities on a MAb it was determined that the CEX process run in bind-and-elute mode was not capable of reducing the amount of dimer present (~2.5%). In an effort to decrease the levels of dimer species we established conditions such that the product impurities bind preferentially over the monomer thus allowing this operation to be run in flow-through mode. There are significant benefits to running CEX in flow-through mode including lower resin costs and higher productivity. Results regarding the optimization of a flow-through mode CEX process are presented in which the amount of HMW species was reduced to <1% and dynamic binding capacity was increased 7-fold with similar levels of residual process-related impurities compared to bind-and-elute mode.
E27 Application of Quality by Design Principles to the Development of a Design Space for a Cation Exchange Chromatography Purification Step Ting Yang, Shire A Quality by Design (QbD) approach was employed to establish the design space of a cation exchange chromatography step for the purification of a therapeutic protein. The most important process parameters were identified by completing a historical review and a risk assessment using failure mode and effects analysis (FMEA). Subsequent experiments, based on a factorial design, were completed to evaluate the impact of selected operational parameters. This study enabled us to quantify the impact of input variables on process performance (e.g. yield) and product quality, thus identifying the design space. Using the identified models, Monte Carlo simulations were then performed to establish in-process ranges for operating and performance parameters.
E28 KANEKA KanCapA, a new mAb purification platform Masahiro Funaki, Kaneka Protein A chromatography has been the first choice of the capture step of monoclonal antibody (mAb) production due to the high purity and high recovery. KANEKA KanCapA is designed for industrial-scale production of mAbs, and is composed of highly cross-linked cellulose and originally designed alkaline resistant protein A ligand. In this poster presentation, performance of KANEKA KanCapA is compared with commercially available Protein A resin. KanCapA shows high dynamic binding capacity and superior elution property, especially for mild acidic pH elution. This is advantage for reducing aggregate formation and high step yield. KanCapA also has excellent alkaline stability that permits 300 cycles of alkaline CIP with 0.1 M sodium hydroxide. KanCapA can be easily packed by flow and axial compression packing methods and shows good and reproducible pressure flow characteristics from pilot to large scale. In conclusion, KANEKA KanCapA shows overall high performance and is considered to be ideal for industrial antibody purification platform.
* This list of posters may not be the final displayed on-site at the event. Changes/additions may occur prior to the event.