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BioProcess International Conference & Exhibition

Conference: October 20-23, 2014 * Exhibition: October 21-23, 2014 * John B Hynes Veterans Memorial Convention Center, Boston, MA

1,500+ Attendees - 160+ Speakers - 150+ Exhibitors

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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 · John B Hynes Veterans Memorial Convention Center · Boston, MA

2014 Poster Presentations

2014 Poster Presentations

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

Updated: September 18, 2014

Analytical & Quality

A1 Determination of the Domain-Specific Degradation Profiles of SpeB Digested Rituximab Biosimilars by Imaged Capillary Isoelectric Focusing (iCIEF) Zichuan Zhang, PPD In this presentation, SpeB digestion coupling with iCIEF was developed for the determination of domain-specific degradation profiles of innovator and generic Rituximab (Rituxan, Mabthera and Reditux). Untreated and base treated (pH 10 at 40 ?C for 24 h) Rituximab samples were digested by SpeB ("FabULOUS", Genovis, Sweden) in reduced condition for 1 h at 37 ?C to obtain Fd, Fc/2 and light chain (LC), and mixed with ampholyte solution consisting of 2% of Pharmalyte 5-8, 2% of Pharmalyte 8-10.5, 0.5% of pI marker 5.85, 0.5% of pI marker 10.45, 0.35 % of methylcellulose and 7 M urea. An iCE3 instrument with UV detection at 280 nm (Protein Simple, CA) was used and Empower 2.0 software (Waters, MA) was used for data analysis. It was observed that untreated Rituxan (GenenTech, US) and Mabthera (Roche, EU) samples shared identical peak distribution and charge heterogeneity, while multiple charge variations of Fc/2 and less acidic Fd species were found from Reditux (Dr. Reddy, India). After 24 h treatment in pH 10, similar degradation products were observed from all three Rituximab samples. These results indicate that the established method can be applied to the domain-specific characterization of biosimilar therapeutic products.

A2 A semi-automated, high-throughput methodology to characterize the N-glycosylation profile of a monoclonal antibody using the Agilent AssayMAP Bravo Elizabeth Dodson, BD Advanced Bioprocessing Protein quality is a primary concern for manufacturers of bio-therapeutics. Protein modifications with greatest impact on therapeutic function are known as critical quality attributes (CQA). The CQA's of a protein can be impacted at several stages of process development including clonal selection, cell culture, protein purification, and final formulation and storage. It is critical for us to have the capacity to assess the impact media and supplements have on protein quality. Here we describe the use of an automated, high-throughput methodology for the purification of monoclonal antibodies from cell culture supernatants with good yields. Further, the same technology was used in the semi-automated isolation and purification of N-glycans with comparable results to a "manual" methodology. The automated methodology allows for purification and characterization of up to 96 samples in under 4 hours as opposed to manual method (5 samples in three days). Taken together, the new high-throughput methodology provides a means of screening large numbers of cell culture conditions for the characterization of protein quality.

A3 Optimization of CHO cell culture medium to target a monoclonal antibody charge variant profile Justin McGrath, BD Advanced Bioprocessing Protein quality of a biological therapeutic can be impacted at several stages of process development including clonal selection, cell culture, protein purification, and final formulation and storage. For BDAB understanding how cell culture media impacts protein quality is of paramount importance. Here we describe a two tiered approach for targeting the charge variant profile of a monoclonal antibody (mAb) expressed in a CHO K1 cell line. An existing animal component free (AF) basal medium did not produce a charge variant profile comparable to the desired profile of a reference protein. As a first strategy the basal media was reformulated to either remove or add certain components. The second approach was a new media screen and DOE around the optimal media with charge variant as one of the guiding parameters. Both strategies resulted in a much improved charge variant profile for the monoclonal antibody.

A4 PetriJet - Innovative lab-automation solution for culture dish handling, imaging and automatic image analysis Felix Lenk, TU Dresden Petri dishes are a user-friendly and easy method for in vitro cultivation of not only plant cell and tissue cultures. Specific growth parameters, productivity and the influence of agar are only a small set of parameters which can be investigated by using petri dishes for cultivation. In larger screening experiments the manual and mostly only qualitative analysis is limited by the number of laboratory staff. Based on the constant development in laboratory automation an innovative solution to these problems is now for the first time readily available even for small and middle scaled laboratories with special requests due to their individual research profiles. With the presented fully automated, bench-top solution towards the handling of culture dishes, the experimental efforts can be reduced significantly and the sample throughput can be increased. For a continuously monitored, systematic, non-destructive and quantitative analysis of the culture dishes an automatic image recognition and analysis has been implemented. Any specific requirement for the analysis can be implemented into the algorithm. For long term experiments every culture dish gets labeled with a QR-code. With this system the culture dish can be tracked immediately by the algorithm and the new results can be displayed with respect to previous images. The precise analysis delivers reproducible results. These might also be used for quality assurance and analytical purposes. The laboratory bench-top device PetriJet can analyze up to 100 culture dishes within one hour. The user interaction for PetriJet handling is limited to starting, loading and unloading the device with a set of 20 petri dishes each. Currently, with dimensions of 800 x 600 x 400 mm the PetriJet is designed especially for small and medium scaled laboratories and reveals an ideal possibility for systematic and quantitatively analysis of samples on culture dishes.

A5 implementation of variable path length technology at Boehringer ingelheim Ramsey Shanbaky, C Technologies, Inc Absorption spectroscopy is used to quantify biomolecules using Beer's Law. At high concentrations, samples must be diluted due to limitation of traditional spectroscopy instrumentation utilizing a fixed path length of 1cm. A gravimetric correction is applied to ensure the accuracy of dilution. The process of diluting, applying the gravimetric correction and washing the cuvette is time consuming and can take several hours in a controlled setting such as Quality Control or Manufacturing. The use of variable path length technology eliminates the need to dilute samples by taking the absorbance measurements at multiple path lengths using a disposable optical fibrettte. The software plots absorbance vs. path length. Using a linear regression, the software calculates a slope for the points and determines the concentration by applying Beers Law. Use of variable path length technology for concentration measurements results in time savings of greater than 80%.

A6 Aggregation of Antibody-Drug Conjugates: The Light Scattering Toolbox for Screening and Characterization Daniel Some, Wyatt Technology Corp. Antibody-Drug Conjugates (ADCs) show much promise as effective therapeutics for cancer and other diseases. However, they often exhibit an increased aggregation propensity compared to their unmodified counterparts due to non-specific interactions arising from attached drug and linker moieties. Light scattering offers multiple techniques for addressing the challenges of formulation screening, and characterizing both aggregates and propensity for aggregation. We present these tools in the context of ADCs and the dependence of aggregation profiles on linker chemistry.

A7 Tracing of Extractables/Leachables through a mAb process using Mobius® ClinicReady Solutions Platform Hyesun McNulty, EMD Millipore The Mobius® ClinicReady Solutions is an innovative single-use platform that provides flexibility to build an entire upstream and downstream process, ranging from buffer /media preparation, clarification, virus filtration and TFF. One of major components of such single use systems is containers (bioreactor and buffer storage bags) which is constructed with polymer based material, hence has potential release of additional extractables/leachables into the final products compared to their traditional counterparts such as glass and stainless steel. These components are gamma irradiated as a mean of sterilization. There is a large body of studies evaluating extractables from various materials, but these studies have insufficient information on how extractalbes are added or removed throughout the process. We conducted a model study to trace extracables/leachables using a typical mAb conditions. A cell culture media (without inoculation) and buffer solutions were processed, collected and analyzed for extractables at nine major process points through Mobius Clinicready solution platform. The results of such study will be presented in this paper.

Cell Culture and Upstream Processing

C1 Case Studies of hERG Membrane Protein and Japanese Encephalitis Virus Production Using a New Innovative Moving Bed Bioreactor Lewis Ho, BioReactor Sciences Human ERG membrane protein has gained great interest as a target for drug discovery. A two fold increased expression of the hERG gene was induced under nutrient limitations. The protein is membrane bound and therefore whole cell recovery from the carriers is required. HEK293 cell was used. Japanese encephalitis virus (JEV) for vaccine has long been the only reliable solution to prevent deadly encephalitis. The virus however is unstable and post-infection conditions for optimal virus production and recovery is critical. We use two case studies to illustrate a new innovative Moving Bed (MB) bioreactor's unsurpassed versatility and functionality to accommodate specific desired conditions and to accomplish the entire upstream processes in one single bioreactor. The mobility of the moving matrix bed allows for the least amount of volume required for seeding, yielding high seed density resulting in increased cell attachment. Similarly, this feature results in high virus infection and DNA transfection efficiency. The MB system facilitates the principle similar to roller bottles intermittently exposing and submersing cells to air and medium to achieve maximum oxygenation with near minimal shear, and also eliminating foaming issues. The moving bed sets a new precedent in performing the mechanism of cell detachment and recovery in place.

C2 A Novel System to enhance the Production of Erythroid Cells from Human Induced Pluripotent Stem Cells using HIF-1α NIHAL ALMURAIKHI, IMPERIAL COLLEGE LONDON Blood shortage is one of the major global concerns as there is a significant imbalance in donations comparing to transfusion demands, which encouraged researchers to develop appropriate substitutes for donated blood using pluripotent stem cells. There has been a rising excitement lately that human induced pluripotent stem cells (iPS cells) could provide patient-specific cells for cellular therapy in addition to their differentiation capability into any cell type, which can be exploited in erythroid cell production. Erythropoiesis is the process of making erythrocytes that can be enhanced by hypoxia-inducible factors 1-alpha (HIF-1α), a transcription factor known to facilitates cellular adaptation to hypoxia by over-expressing specific genes and stimulating many metabolic processes, including erythropoiesis, and angiogenesis. In this study, we have established a novel protocol to generate erythroid cells from human iPS cells using HIF-1α as a key enhancer. Beside the use of the standard cytokine cocktail used for erythroid induction; Epo, SCF, FLT3, TPO, IL3, and IL6, other growth factors were used; BMP4, VEGF, FGF, in addition to 5% serum. Supplementing the cells in 2D culture system with our novel optimized concentrations of the said cocktail under hypoxic condition showed the highest yield erythroid markers, mainly CD235a. Further maturation of those cells is required in order to achieve fully mature and functional RBCs phenotype. These results must be supported by the detection of Rh type and ABO grouping to ensure the presence of RBCs antigens. Eventually, after optimizing an enucleation protocol, globin detection and O2 dissociation curve must be also made to ensure functionality of the hemoglobin (incomplete work). Thus, considering all the above, the ultimate aim of this study is the efficient production of mature and functional RBCs in vitro from patient-specific iPS cells using HIF-1α to facilitate erythroid progenitor maturation and proliferation.

C3 An Automated Bioreactor Sampling Solution for Assuring On-line PAT Analytical Fidelity Michael Biksacky, Flownamics, Inc. In the current bioprocessing environment a significant unmet need still exists for automated, precise, real-time analysis of important culture process parameters such as nutrients, metabolites and various cell measurements. Transforming traditional off-line analytical methods into effective, automated, on-line process analytical technology (PAT) platforms requires rapid and precise analysis that performs as good as or better than the manual off-line analytical method. An automated, on-line bioreactor sampling system was evaluated for its ability to preserve the analytical integrity of various integrated 3rd party off-line analyzers typically used for culture process monitoring. The performance evaluation encompassed a broad range of analytes and measurement ranges, while comparing automated on-line analysis with the analyzer’s respective manual, off-line analytical method. In short, the analytical fidelity of each integrated analyzer was preserved by the automated on-line sampling system, which demonstrated a reliable, automated on-line PAT solution for real-time bioreactor culture monitoring and control.

C4 Seg-Flow® Automated Nutrient Monitoring and Continuous Feed Platform for Precise Nutrient Feed Control Ashley Fisher, Flownamics, Inc. Fed-batch culture processes are commonly used for the production of various biotherapeutic and other industrial commodities. A widely employed nutrient feed control strategy for both cell culture and microbial fermentation processes is direct feedback control, in which the nutrient feed is directly controlled by the nutrient, i.e., glucose, concentration. However, various iterations of this feeding approach exist which may not be conducive to culture growth and productivity. In order to optimize culture performance, the means by which the substrate is fed to the culture is just as, if not more, important as maintaining the desired media substrate levels. An automated feedback control platform, which employs a continuous feed strategy based on the culture's real-time nutrient consumption rate, has been developed by Flownamics®. This integrated platform consists of the Seg-Flow® automated on-line sampling system, a nutrient analyzer and a feed pump. The Seg-Flow system was evaluated for its ability to precisely control media glucose concentrations in both low and high consumption rate culture simulation models. The results showed that the integrated Seg-Flow system was able to precisely control media glucose concentrations at the prescribed levels through real-time glucose consumption rate calculation and continuous feeding.

C5 High Throughput Screening for Mammalian Cell Clones Wen Wang, Massachusetts Institute of Technology For the past decade, biopharmaceutical industry had a fast growth, creating a hundred billion dollar market worldwide with the approval of recombinant proteins, monoclonal antibodies and nucleic acid-based drugs. Among all these production systems, the Chinese Hamster Ovary (CHO) cells are the most widely used mammalian cell systems for transfection, expression, and large-scale recombinant protein production, due to its advantages in accurate glycosylation for production of natural proteins in vitro. In recent CHO cell culture technology, significant improvement in recombinant protein production leading to a titer up to 10 g/L was achieved, to meet the high market demand by using stable and high producer together with the optimization of culture process. However, rapid selection for high production of clones is still needed. In current study, we apply a new screening concept for on line and in situ high-throughput screening for mammalian cell system. CHO cell line producing anti-her2 was used as the model mammalian cell system. By measuring the key parameters of the cell culture system on line, the performance of cell line can be predicted, and the high producers can be identified without performing invasive assays. The impact of the research will be very significant in upstream bioprocesses. In cell line development, product titer is a crucial parameter for clone selection. Our method does not require any external reporter, and it is a non-invasive liquid-based assay. We only need to monitor basic information of cell culture in order to calculate the product quantity. High and low producers can be easily identified in a high-throughput manner on-line and in situ using automated handling and aforehand programming. This will be a revolutionary change to current mammalian cell screening system. Keywords CHO Cells, High Throughput, Fast Screening High Producer, Product Prediction

C6 Perfusion CHO Cell Culture in BioBLU® 5p Packed-bed Single-use Vessel Nick Kohlstrom, Eppendorf The market for secreted recombinant protein therapeutics, including blockbuster drugs in the form of humanized monoclonal antibodies (hmAbs), has become a multi-billion dollar industry with the expectation of continued growth. One of the most cost effective methods for the production of secreted proteins is the packed-bed vessel operated under perfusion conditions. The maximum cell densities achieved in a packed-bed vessel is typically much higher than suspension cell culture or microcarrier-based adherent cell culture. The protein harvest can be carried out continuously, providing unparalleled product yield. This poster provides an example of using a BioBLU 5p packed-bed single-use vessel to conduct perfusion CHO cell culture producing a secreted hmAb. The BioBLU 5p vessel (pre-loaded with Fibra-Cel® disks) was controlled by a New Brunswick™ CelliGen® BLU benchtop bioreactor. The BioBLU 5p vessel was inoculated at an initial cell density of 0.3 x 10^6 cells/mL. Fourteen days of perfusion cell culture was conducted with a working volume of 3.75 L. Glucose, lactate and hmAb concentration were monitored daily. The glucose consumption rate was used to estimate the cell density in the packed-bed vessel. After 12 days, the culture reached to a peak cell density of approximately 10 x 10^6 cells/mL.

C7 Large-scale Production of Human Mesenchymal Stem Cells in BioBLU® 5c Single-use Vessels Khandaker Siddiquee, Eppendorf, Inc. Stem cell-based regenerative medicine has great potential to revolutionize human disease treatments. Among the various stem cell platforms, mesenchymal stem cells (MSCs) represent one of the highest potentials as evidenced by clinical trial activities. Currently, there are over 400 clinical trials based on MSCs registered at clinicaltrials.gov. Although successful expansion of MSCs in vitro has been well established, the large clinical-scale production of MSCs remains a bottleneck, potentially limiting the immediate clinical applications should some of the ongoing trials receive FDA approval. In this study, we demonstrate the success of large clinical-scale culture of human adipose-derived mesenchymal stem cells (AdMSCs) in an industrial single-use vessel at 3.75 liter (L) scale (working volume). The vessel offers a precision controlled environment for the ideal growth of stem cells under simulated physiological conditions. Stem cells and culture media were monitored, analyzed, and controlled, thus allowing us to produce AdMSCs in large clinical-scale quantities while maintaining healthy stem cell properties as evidenced by stem cell marker assays and differentiation assays performed at the end of the culture. Furthermore, with clinical relevance in mind, every cell culture step from T-flask to shake flask to bioreactor vessel was conducted strictly using single-use consumables.

C8 Particulates in Single-Use Bags Mike Johnson, Entegris The utilization of single-use systems in downstream applications is rapidly increasing. With this increase comes a heightened concern about particle contamination and the associated potential for health and safety risks. Particles found in single-use systems can be traced to several sources, including the system's materials of construction, its physical attributes and the environment in which the product is made. Particles found in drug products can have real patient implications which may include reduced drug efficacy or the blocking of blood vessels resulting in tissue or internal organ damage. The risk associated with particles depends upon several factors including the size, shape and quantity of particles present; the composition of the particles; the physical attributes of the patient; and the route of drug delivery. In order to better understand the impact of single-use systems on the particle content of drug products, we evaluated six different single-use bags from five different manufacturers, quantifying the number and size of particles present in each. This presentation provides the results of our evaluation, along with a comparison of the data to the limits established for USP <788> Particulate Matter in Injections.

C9 Inclusion of a Micro bioreactor system in the MaxXpress platform for Cell Line & Process Development Jason Rahal, Cobra Biologics ltd Cobra Biologics is an established manufacturer of monoclonal antibodies, other recombinant proteins, DNA and virus. Over the last 15 years we have supplied more than 320 batches to more than 100 customers located in 19 different countries on all continents of the world. The need for rapid cell line and process development push processes to higher throughput and smaller scale. Tight time lines and development costs are crucial when developing a platform approach for antibody production, from cell line development to GMP manufacture. Evaluation of cell culture media and additives, as well as process parameter optimisation, can be very time consuming. An automated, disposable micro bioreactor system was included in Cobra's maxXpress platform. This was to provide an approach where cell culture media and additives can be tested while maintaining bioreactor process parameter control, thus facilitating subsequent scale up of the fermentation process.

C10 Enhancing cell culture process and overcoming challenges for antibodies utilized in site-specific conjugation Takuya Kikuchi, Agensys Possessing both the efficacy of cytotoxic drug and tumor selectivity, antibody-drug conjugates (ADCs) have been emerging as a new class of anticancer therapeutics. Traditional lysine- or cysteine-based conjugation technologies generate heterogeneous ADC molecules, which exhibit different pharmacology and present challenges to controlling safety, as well as manufacturing and analytical characterization. The site-specific ADC technology we are using produces ADCs with the same drug-antibody ratio. While homogenous ADCs are beneficial, in some cases a significant challenge associated with this technology is the low expression of antibody protein. In this work, we investigated how feed media components and cell culture processes affect protein expression and cell growth. In particular, we found that some feed media components strongly inhibited antibody production. By optimizing the feed medium and the cell culture process, we improved antibody titer by 75% compared to the original process.

C11 An Integrated Microbial Expression Toolbox to Enable QbD Process Development April Stanley, Cytovance Biologics, Inc. The Gram negative bacterium, E. coli, is a true workhorse in the biopharmaceutical industry and has a well-established track record of FDA approval and commercialization. Despite its broad utility at the bench, many commercial expression systems and hosts pose risks and obstacles in terms of cost of goods, scale-up challenges and in-process control. Researchers often lack the manufacturing expertise or understanding of E. coli genetics to design out the problems before they arise. Cytovance has developed a new microbial expression toolbox that works for manufacturing. Our integrated approach from DNA to DOE will be presented along with a case study.

C12 Impact of raw material variability on glycosylation profile of a CHO-derived humanized monoclonal antibody Jayanthi Lakkyreddy, Genentech Variability and a shift in glycosylation profile were observed during process development for a CHO-derived humanized monoclonal antibody. This shift was attributed to the change in raw material lots, specifically ferrous sulfate (FeSO4). The trace metal manganese (Mn) can be an impurity in FeSO4, and is a co-factor for many of the enzymes involved in the glycosylation pathway. It was observed that some cell lines were more sensitive than others to minor changes in the concentration of trace elements such as iron. For such cell lines, the presence of trace element impurities in raw materials poses a risk for maintaining consistent product quality. Studies were performed to overcome impurity-driven variability in glycan profile by increasing the target Mn concentration in the production media. The results from these studies showed that the glycan distribution was more consistent when the Mn concentration was increased to levels above the impurity range coming from the FeSO4 raw material.

C13 MANUFACTURING OF A CHO-DERIVED MONOCLONAL ANTIBODY - A SCALE-DOWN APPROACH FOR A SENSITIVE CELL LINE THROUGH BIOREACTOR DESIGN AND CONTROL PARAMETER MODIFICATIONS Delyan Rusev, Gilead Sciences Scale translation of monoclonal antibody (mAb) manufacturing processes often presents an operational challenge due to the fact that many volume-dependent parameters scale non-linearly, and cannot be matched across bioreactor volumes. For this reason, small scale or pilot scale performance may not translate directly to large scale bioreactors, resulting in lower productivity and differences in growth and metabolite profiles. For a monoclonal antibody process using a sensitive CHO cell line, differences in growth, metabolic, and control profiles were identified between manufacturing scale and the small scale model originally used to develop the process. Various bench top bioreactor modifications (configuration and control strategies) were evaluated in order to mimic the large scale performance. A final scale-down model addressing multiple hydrodynamic shear parameters, such as bubble size, mixing rate and bubble distribution was developed to effectively reproduce selected performance attributes of the large scale production culture. The scale-down model was then used to assess potential process improvements that would optimize the performance of the large scale bioreactors.

C14 Cell Culture Process Development for Fed-Batch Using a Novel Nutrient Additive Shawn Barrett, Thermo Fisher Scientific Bayer CHO cell-based process development for mAb production across 3 different host cell lines and 7 different projects has been summarized. A novel nutrient additive was extensively evaluated in 6 out of the 7 projects. By implementing the new platform process, high productivity was easily achieved in addition to desirable process attributes. The simple platform process also enabled the rapid transfer of antibody projects into clinical manufacturing and was demonstrated to be a robust, reproducible, and GMP-friendly fed-batch process.

C15 VAYU meter - Bio monitoring device Moria Shimoni, VAYU sense VAYU sense's rapid screening device, the "VAYU meter", has been developed for the detection of living cells and microorganism's in growth medium. The VAYU meter measures CO2 metabolic gas concentrations produced during cell's respiration and proliferation. VAYU meter enables continuous metabolic gas detection for high sensitivity bio-process control, monitoring the growth rate and bio concentration. By means of IR absorption, our device allows in-situ detection of cells and microorganisms in fermentors or bioreactors directly throughout the reactor exhaust without the need for opening, sampling or incubating. This device can be applied for quantitative analysis and estimation of a level of biological activity of cells and microorganisms in pharm processes. Fermentation processes of most important and complex pharmaceuticals require precise culture base growth. From the early stages of commercial proteins production, the handling of cultures is being subject to challenges. Commercial production of products on a large scale depends largely on the stable maintenance of cell level. To optimize biotech production processes and improve yield and economics there is a need to achieve better, precise, real time process understanding and improved equipment utilization. The VAYU meter is a real-time process status monitoring solution capable of accurately indicating relative reaction conditions. This high resolution continuous detection system is successful in detecting cells and microorganisms' concentration. VAYU meter - Advantages and Features ? Enables to control culture growth throughout all stages of the fermentation process ? Contact-less and easy to install on every system (fermentors; bio-reactors, etc.) ? Real-time monitoring of biomass production process ? Shortens R&D stages ? Optimized production processes ? Improved yield

C16 Evaluation of the BioFlo® 320 process capabilities Ma Sha, Eppendorf Inc. Historically, stirred-tank fermentors and bioreactors have been the trusted design for cultivating all types of submerged cultures including suspension and anchorage-dependent mammalian, insect, yeast, plant, and microbial cultures. The tried and tested tank design offers scalability and proven reproducibility which is pivotal for cost-saving process development and productivity. However, for the most part, the glass or stainless steel autoclavable systems and the single-use equipment have remained separate. In order to meet the demand of improved process flexibility and cost savings, Eppendorf recently developed a new bioprocess controller that combines the capabilities of autoclavable and single-use systems into one advanced bioprocess control station, the BioFlo 320. The newest offering in the Eppendorf bioprocess portfolio, the BioFlo 320 blends design and utility in one all-inclusive package. An industrial controller design, interchangeability of autoclavable and single-use vessels, intelligent sensors, Ethernet connectivity, and an enhanced software package are only a few of the features that set it apart from the competition. This poster characterizes the BioFlo 320's capabilities in terms of key process parameters including oxygen transfer rate (OTR), volumetric mass transfer coefficient (kLa), water and energy consumption, tip speed, specific power input, and Reynolds number. These data allow a quantitative analysis of bioprocess engineering parameters, thus allowing potential users to gain insight into the system's high density cell culture and fermentation capabilities.

C17 Continuous CHO Cell Culture Perfusion Platform for Process Development and Manufacturing Olivier Berteau, Charter Medical, Ltd. Bioreactor design is a complex engineering task requiring fine-tuning of bioreactor design and operational configuration to enable the cultured organisms to achieve their desired function. Before large-scale commercial manufacturing can be implemented, it is typically necessary to scale up a bioreactor during a process development stage. However, conventional scale-up techniques are fraught with technical challenges and financial risks. Perfusion bioreactor system in which a plurality of modular perfused bioreactors is operated in parallel to produce and maintain a biological cell culture is presented. In order to prevent contamination and facilitate segregation of particular peripheral bioreactors, the distributed system is configured such that one-way fluid communication is established from the central bioreactor to each of the peripheral bioreactors while maintaining fluid isolation among the peripheral bioreactors. In accordance with one exemplary non-limiting embodiment, a distributed perfusion bioreactor system is provided for producing and maintaining a continuous biological cell culture. The modularity of the peripheral bioreactors enables a scale-up free design of an exemplary bioreactor system that avoids the disadvantages of a conventional scale-up technique. The modularity of the peripheral bioreactors enables detection of process deviations and contamination, and easy and efficient segregation or restart of an affected peripheral bioreactor branch without disrupting the entire system. A distributed bioreactor arrangement is presented to maintain the cell culture continuously for extended periods of time using a commercially available CHO cell line.

C18 A new DO control strategy of mimicking large scale DO heterogeneity to improve the cell culture scale down model Weimin Lin, Biogen Idec Oxidative stress is caused by elevated production or reduced detoxification of reactive oxygen species (ROS) in cells. ROS can directly damage the cells and is associated with cell death. When scaling up a process from bench scale 3-L bioreactors to 15000-L bioreactors, some cell lines were observed to have more than 50% growth decrease. The hypothesis is that the localized high DO exists due to mixing heterogeneity at large scale. To advance understand and control our bioprocess, we developed a scale-down model based on the impact of high DO using a new DO control strategy at small scale to mimic large scale DO heterogeneity, resulting in performance that is similar to large scale experience. The improvements to the cell culture scale down model and future work will be described.

C19 Maximize without Compromise: Using super-concentrated feeds in fed-batch cell culture Becky Moore, Thermo Fisher Scientific High performance commercial Chinese Hamster Ovary (CHO) protein expression systems require fed-batch processes that maximize bioreactor working volume, maintain specific productivity (i.e., product per cell), and provide sufficient nutrient delivery to maintain proper protein quality. The challenge in media development is to overcome traditional solubility limitations that impacts initial bioreactor and feed volumes causing product dilution and compromised bioreactor yield. The development of this new technology enables simple, pH neutral, super-concentration of feeds up to 200g/L, which permits either reduced product dilution via feed volume or increased titers with additional feeding as desired. Recent studies using a CHO-K1 cell line demonstrated that after super-concentrating the feed by 2x, additional feed supplement could be added to the bioreactor resulting in a high performance fed-batch process exceeding 7g/L yield. This study highlighted manufacturing advantages including single-part water-only reconstitution, higher titers due to the use of this super-concentrated format, higher volume bioreactor inoculum, and flexibility in bioreactor usage. Incorporation of this technology into fed-batch bioprocesses enables flexibility in bioreactor start volumes and feed volumes, minimizes risk associated with multi-part extreme pH feed strategies, and allows for superior culture performance in recombinant biotherapeutic drug manufacturing.

C20 Determining Agitation Requirements for Microcarrier Processes: Method Development in the Mobius CellReady 50L Single-use Bioreactor Kara Der, EMD Millipore The Mobius® CellReady bioreactors have been demonstrated to provide a scalable platform for optimal suspension cell culture performance. However, not all cells can be grown in suspension. Adherent cells require a solid surface upon which to grow and have traditionally been grown using 2D tissue culture flasks or roller bottles in which scale-up becomes cumbersome. Alternatively, adherent cells can be grown in 3D suspension culture if a solid surface, such as a microcarrier, is provided. In order to insure successful cell culture, the microcarriers must be kept in suspension throughout the duration of the process. A methodology for determining the minimum agitation rate required to maintain adequate microcarrier suspension and thereby avoid microcarrier settling has been developed using collagen coated microcarriers (SoloHill) in the Mobius CellReady 50L bioreactor. We have demonstrated that by maintaining a constant agitation rate it is possible to maintain a similar suspension environment as the volume of the process is increased as long as the microcarrier concentration remains constant. Additionally, we have employed the Zwietering correlation to predict, and experimental analysis to verify, the agitation rates required to maintain suspension of different concentrations of microcarriers.

C21 Development and characterization of large scale production of a recombinant rabbit monoclonal IgG Bill Zeck, Abbott Development of a robust immune response to small hapten immunogens is relatively difficult to obtain in mice compared to rabbits. We immunized rabbits with a vitamin-D immunogen and isolated six rabbit monoclonal antibodies directed against 25-OH vitamin D. All monoclonals exhibited similar binding characteristics despite divergent sequences within their respective complimentarity determining regions (CDRs). Cell culture process development and downstream purification processes were developed to ensure high levels of production of the recombinant IgG from a stable CHO cell line. Potency testing of three large scale pilot production runs revealed equivalent performance thereby demonstrating the robustness of the processes and their readiness for full factory implementation.

C22 Effects of Cell Density, Aging and Media Formulation on CHO Antibody Expression: Feasibility of Titers >1 g/L. Weili Wang, MaxCyte, Inc Flow electroporation provides a means of fully scalable, highly efficient transient gene expression (TGE) that results in antibody titers >1 g/L and production of multiple grams of antibody from non-engineered CHO-S cells. Pre-transfection cell health, growth, and passage number can affect transfection efficiency and cell viability levels and, thus, antibody production. In this poster, data using flow electroporation via the MaxCyte STX® Scalable Transfection System are presented that examine the effects of pre-transfection parameters such as, media supplementation, cell density, and cell passage number on CHO-based antibody production. In conjunction, data illustrating the effects of post-transfection parameters such as seed density, media formation, and feed strategy are shown. Taken together these data will demonstrate the ability of MaxCyte STX electroporation to i) produce secreted antibody titers >1 g/L yielding multiple grams of antibody within two weeks of a single CHO transfection, ii) scale-up without protocol reoptimization while maintaining transfection performance, and iii) enable fine-tuning of pre- and post-transfection parameters to balance speed, cost, and antibody yield. In summary, these data will support the use of flow electroporation via the MaxCyte STX as an enabling technology that ultimately reduces early-stage labor and cost expenditures, while delaying the migration to stable cell line generation by extending the use of CHO-based TGE through mid- to late-stage antibody development.

C23 Alternative Splicing and Method for Specific Detection of Splicing Variants in CHO-K1 Cell-line during Fed-batch Production Process Zhixin Shao, Roche Diagnostics Alternative splicing is a process by which the exons of the RNA produced by transcription of a gene are joined in multiple ways during RNA splicing. The resulting different mRNAs are usually translated into different protein isoforms. In this way, a single gene may code for multiple proteins. Amyloid precursor protein (APP) is an integral membrane protein expressed in different cells/tissues in many organisms. It has been previously reported that Chinese hamster ovary (CHO) cells express full length APP. The biological function of APP in CHO cell-line remains unclear. Currently no detailed information about CHO APP gene structure/expression in K1 cell-line is yet available. In addition, no alternative splicing isoforms of APP have previously been detected in CHO cells. In this study, novel splicing test methods based on amplifying and sequencing APP mRNA isolated from cell samples have been developed. Described is the identification of at least four (4) different splicing variants of the APP mRNA in CHO-K1 cells. These mRNA variants occur mainly through skipping one or more full exons within the APP gene. These methods allow for improved detection limits of spliced RNA variants in cell samples particularly from a fed-batch production process.

C24 Gas Analysis Mass Spectrometry Applications in Fermentation and Cell Culture Processes Pete Traynor, Thermo Fisher Scientific As a PAT tool, on-line analysis of the inlet and outlet gases of sparged fermentors, provides an ideal means of characterizing the fermentation state. Key indicators include the Carbon Dioxide Evolution Rate, Oxygen Uptake Rate and the ratio of these two parameters: the Respiratory Quotient (RQ). These measurements are used to monitor and control the process: for example to decide when to transfer a seed tank culture to inoculate the fermentor, to control sparging gas flow rate and composition, to detect contamination by foreign microorganisms and to control the substrate feed introduction rate. They are also used to determine the cell density, to compare with historical trend data to verify the process is proceeding normally, and to determine the completion of the batch. Process Mass Spectrometry is the most powerful technique for gas analysis of fermentations. The multi-component measurements are fast and precise. It has a wide dynamic range to allow measurements over the range of ppm to percent and also the sensitivity, stability and resolution to determine minute changes in concentrations. A mass spectrometer for fermentation is usually supplied with a multi-point sampler so that multiple samples are measured with one instrument. As an example, with typically 10 seconds being required per sample point, 60 sample points can all be measured with a cycle time of 10 minutes on one instrument. Additionally Process Mass Spectrometry has the flexibility to be able to monitor many different components. This poster describes the technology and benefits of fermentation monitoring by Process Mass Spectrometry.

Formulations/Delivery

F1 QbD Guided Process Development of a Novel Cationic Nanoemulsion for Delivery of Nucleic Acids Sonia Gregory, Novartis Vaccines Emulsions have been used to enhance potency of a vaccine or in some cases for delivery of an antigen. Here we discuss the development of a novel Cationic Nanoemulsion (CNE) for delivery of nucleic acid-based vaccines. CNE has been shown to be capable of safe and efficacious delivery of a 9 kb self-amplifying mRNA in small animal and non-human primate studies(1). CNE was developed based on the Novartis proprietary MF59 oil-in-water emulsion adjuvant. The development and scaled up production of CNE was guided by Quality by Design principles. The critical quality attributes were assigned to the product and assessed during each step of the process to define criticality of process parameters. Upon identification of critical process parameters, the studies to better define operating ranges were executed. Overall the use of QbD approach has eliminated redundancy and improved efficiency during the development of CNE process. (1.) Brito, L.A. et al. A Cationic Nanoemulsion for the Delivery of Next generation RNA Vaccines. Molec. Ther. 19 Aug 2014 10.1038/mt.2014.133

Manufacturing

M1 Sensitivity Analysis of BioGTL Process Qiang Fei, National Bioenergy Center In light of the relatively low price of natural gas and increasing demands of liquid transportation fuels, attention has begun to turn to novel biocatalyst for conversion of natural gas into diesel fuel (Bio-GTL). A techno-economic analysis (TEA) was performed for an integrated process using fermentation process converting natural gas to microbial lipids and catalytic upgrading lipids to diesel blend stocks. A sensitivity analysis was carried out based upon the identified key parameters, such as plant capacity, process technology (yields and rates), productivity, and raw material cost. The sensitivity analysis cases were also presented to compare the production of natural gas-derived diesel with different fuels produced from various platforms.

M2 Microalgae Culture Using the DASGIP PBR4 Module for Illumination with a New Brunswick CelliGen 310 Bioreactor Stacey Willard, Eppendorf, Inc. The number of bioprocess applications for microalgae has increased in recent years, particularly in the field of biofuel production. The global biofuels market is predicted to reach US$100 Billion in 2014. As a result, microalgae bioprocess applications have the potential to expand into a multi-billion dollar sector of this growing global market. The combination of the New Brunswick CelliGen 310 benchtop autoclavable bioreactor and the DASGIP LED Illumination System creates a bioprocess setup which is capable of supporting high density microalgal growth. Using the stand-alone DASGIP PBR4 Module, LED illumination spectra and intensities can be controlled for optimal support of all types of chlorophylls and carotenoids. For this study in which high density culture of up to 1.5x107 cells/mL was achieved, the unicellular marine alga, Dunaliella tertiolecta, was used.

M3 High cell density fermentation of Escherichia coli using the New Brunswick™ BioFlo® 115 Benchtop Fermentor Bin Li, Enppendorf, Inc Escherichia coli (E. coli) is a Gram-negative bacterium that has had a long history in the world of laboratory and industrial processes due to its ease of manipulation and well understood genome. It is widely cultured under aerobic conditions. High cell density cultivation of E. coli is a powerful technique for the production of recombinant proteins. Indeed, 30 % of the FDA-approved biopharmaceuticals on the market are produced in E. coli. The New Brunswick BioFlo 115 from Eppendorf is an easy-to-use benchtop bioprocess system, with built-in controls for operation as a microbial fermentor. Featuring Rushton impellers, high speed direct drive motor, and optional thermal mass flow controller (TMFC) and automatic gas mixing, this system is ideal for use in R&D labs, universities, teaching facilities, testing labs and more. This poster presents a successful example of high density fermentation of E. coli using a BioFlo 115 fermentor with 2 L working volume heat-blanketed glass vessel. E. coli cultivation achieved a high optical density (OD600) value of 140 at 11 h without optimized fermentation medium and conditions.

M4 Successful Implementation of Automation in Single-Use Bioprocessing Todd Kapp, Parker domnick hunter Single-use bioprocess technology has become near ubiquitous within biologics manufacturing facilities. Benefits such as the avoidance of product cross contamination, flexibility process configurations and reduced capital expenditure requirements are especially attractive to contract manufacturing organizations. Single-use processing steps can be manual, semi-automated or fully automated. Many biomanufacturers are still evaluating the level of automation that is appropriate within their operations before making facility investments they will need to live with for years to come. This presentation will look at a project to automate a single-use, final bulk filtration and dispense processing step at Fujifilm Diosynth Biotechnologies, a biopharmaceutical contract manufacturing organization. A key component of the project was to define the level of automation that was appropriate for Fujifilm Diosynth Biotechnologies in order to deliver the required benefits of process control and consistency, increased speed and operational flexibility.

M5 The Quincy College Biotechnology and compliance training program Isso Bayala, Quincy College With strong industry support, Quincy College has developed a Biotechnology and Compliance Program for training in bio-pharmaceutical manufacturing. Students are trained on both single-use (disposable) and traditional stainless steel bioreactor systems while following Good Manufacturing Practices (GMP) in a fully equipped, state-of-the-art facility. The program prepares students for entry level positions in biomanufacturing, quality control, quality assurance, and biomedical research. Short courses in numerous areas of biomanufacturing are offered for incumbent worker training. In addition to hands-on laboratory courses, students train on a highly interactive, comprehensive web based e-learning environment to practice industrial scale single-use biopharmaceutical manufacturing. Employment rate for students who have successfully completed the program is ninety percent.

M6 Innovative Pre-Packed Process Columns for Biomanufacturing Lynn Sutherland, Atoll Bio Greater adoption of single-use and disposable systems in biomanufacturing and development will require improved downstream device innovation, particularly for pre-packed chromatography columns. Atoll developed and evaluated a disposable, pre-packed, pre-qualified column platform, MediaScout® MaxiChromAC. MaxiChromAC is suited for the purification of biopharmaceutical compounds such as mAbs and vaccines in preclinical, clinical phase I and II studies. The column can also be used in full scale manufacturing depending on the scale and titer of the fermentation process. The innovative design of the column allows direct connection to state-of-the-art disposable chromatography systems. It also allows the packing of a broad range of commercially available chromatography media of different particle sizes including rigid, semi-rigid and soft polymeric backbones. The transparent column housing allows top to bottom bed inspection during the chromatographic workflow. The maximum working pressure is up to 6.0 bar. This outline shows a complete packing evaluation for a 14.1 L pre-packed MaxiChromAC column including HETP and Asymmetry measurements before and after ground and aircraft transportation. Furthermore, a case study for a protein purification process was performed and compared to standard stainless steel equipment.

Recovery & Purification

R1 The Renaissance of Expanded Bed Adsorption? Martin Hoffman, Biotechflow 20 years after development, Expanded Bed Adsorption chromatography fell out of favour in Bioprocessing. Proposed improvements in process efficiency- obtaining high yields from unclarified feed stocks- were compromised by column design and process run failures; in part caused by blocked meshes, lipid breakthrough and demand for high buffer volumes producing high eluting volume. Current media optimize Dynamic Binding Capacity within the expanded bed. Coupled with improved inlet design, increased bed densities are achieved, which permit high flow rates (450 cm/hr) and throughputs. Direct product capture reduces unit costs by 66%, and labour cost and process time. Comparisons with packed bed chromatography result in higher product yield and 43% reduction in buffer volume. Fully-scalable, cGMP EBA column design improvements prevent vortex formation, whilst ultrasound technology enables contactless monitoring of process dynamics and flow rate via feedback to the skid; both factors maximize expanded bed stability. Reduced column footprint and height requirements increase versatility. In recent years, step changes in incubation techniques, media design, column technology and sensor applications have enabled proven improvements in EBA process efficiency and operations. The initial promise may now be considered as surpassed, and a renaissance of Expanded Bed Adsorption Chromatography should be proposed. (200)

R2 Improving Safety and Efficiency in a Demanding Pilot Plant Environment Ron Massicotte, Genentech / Roche Illustrate & explain several improvements that have enhanced safety and efficiency in a busy pilot plant environment.

R3 Nanofibers for high productivity downstream processing Oliver Hardick, University Ciollege London • Payers worldwide are becoming ever more price sensitive and successful exploitation of the emerging and biosimilar markets will require a rethink of the current production strategies to re-focus on cost of goods, flexibility and 'developability'. • Chromatography is moving from a 'one size fits all' bead based process to one of increasing product stratification as new entrants launch technically specified products to address specific process issues.

R4 Direct Quantification of Residual Host Cell DNA Nancy Ngo, Bio-Rad Laboratories Many therapeutic proteins and vaccines are manufactured using bacterial and mammalian host cells. Manufacturing processes are prone to leaving biological impurities from these cells, such as host cell DNA (HCD). The presence of HCD in drug substances poses safety concerns and must be removed to ensure product quality and safety. Regulatory agencies, such as the U.S. Food and Drug Administration (FDA) and the World Health Organization (WHO), have provided guidelines defining acceptable levels of HCD allowed in final drug products; the upper limit is 100 pg/dose and 10 ng/dose as stated by the FDA and WHO, respectively. Therefore, the method to quantify residual HCD and monitor DNA clearance should be highly sensitive to meet these regulatory requirements. Here, we introduce a highly precise and sensitive method for residual HCD quantification, without the need for DNA extraction, using droplet digital PCR (ddPCR). We created a panel of test matrices to simulate various process intermediates and analyzed these samples using ddPCR. Spiked-in DNA can be fully recovered even in complex matrices with high precision and femtogram-level sensitivity. The results from our study clearly show the effectiveness of ddPCR in detecting residual HCD by a direct method and without the need for DNA extraction.

R5 An Optimized Approach for Anion Exchange (AEX) Chromatography to Enhance Productivity and Decrease Cost of Goods Yuyi Shen, XOMA(US)LLC Yuyi Shen*, Jimmie Pagsolingan, Jeffrey Bettencourt, Shelly Parra *Corresponding author Abstract: Anion-exchange (AEX) products (resin/ membrane adsorbers) are commonly used as a monoclonal antibody purification polishing step in process flow-through (FT) mode for reduction/removal of process impurities such as DNA, host cell proteins, endotoxins . Using a high-performance AEX resin at high operating flow rates and shorter bed heights, can significantly increase throughput while providing robust impurity clearance and high yields. Combining this approach with the convenience of pre-packed column will provide a more productive unit operation within the manufacturing process, reducing cost and time. The poster will show how combination of AEX resin and utilization of pre-packed and pre-qualified column can drive significant reductions in process time and costs compared to the traditional approach of in-house packing and qualifying the column for purification of monoclonal antibodies.

R6 Characterization of a newly introduced salt tolerant anion exchange resin useful for process scale chromatographic purification of proteins and monoclonal antibodies Kelly Motter, Tosoh Bioscience Conventional Anion Exchange Chromatography (AEX) resins containing quaternary amine or DEAE ligands, and generally used in bio manufacturing as a purification step, require low to moderate ionic strength to achieve sufficient binding and display reduced or zero binding capacity for proteins in relatively high salt concentrations typically associated with post-protein A purification monoclonal antibodies (mAbs) or undiluted biological feedstock. Because of this, dilution becomes necessary which leads to both loss of time and an increased cost of operations. Tosoh has introduced a new, salt-tolerant AEX resin, TOYOPEARL NH2-750F, consisting of a polymeric bead (45 µm particle size and > 100 nm pore size), functionalized with a primary amine ligand. BSA begins to elute at a concentration of approximately 1.0 mol/L NaCl compared to 0.14 - 0.40 mol/L for other AEX resins. In addition, the DBC of BSA approaches 70 g/L across a range of pH values and conductivities, allowing for excellent alkaline stability. Here we discuss the characterization of this resin for a variety of process scale applications throughout the mAb purification process, beginning with the capture of proteins from biological feedstock (mammalian cell culture, plasma, bacterial feedstock, etc…) without dilution and continuing through intermediate or final purification process steps.

R7 CaptureSelect™ technology: Introducing one-step selectivity in the purification of biological products Frank Detmers, Thermo Fisher Scientific Affinity chromatography is one of the most effective methods for purifying protein therapeutics. The CaptureSelect™ ligand technology addresses protein purification challenges and provides a platform approach by introducing a highly selective capture step for primary recovery purification. These solutions offer unprecedented specificity to the target protein ensuring mild elution conditions for sensitive proteins at any scale and independent from the feedstock. Author: Dr Frank Detmers, Director Ligand Application, Bioproduction, Thermo Fisher Scientific

R8 The Benefits of High Performing Chromatography Resins Including POROS® XQ, A New Strong Anion Exchanger Christine Gebski, Thermo Fisher Scientific POROS resins offer unique performance attributes and drive benefits to downstream processing. POROS XQ is the newest addition to our product portfolio. This high capacity, salt tolerant, high resolution anion exchanger enables unique selectivity, higher product yields through better separation and reduced cost of goods. Applications data will be used to demonstrate the benefits of POROS resins to capture and polish chromatography and process modeling will be used to demonstrate cost of goods improvements and process efficiencies that can be realized.

R9 Maximizing Viral Clearance for High Titer Validations Alexander Schwartz, Asahi Kasei Bioprocess Virus filtration is considered to be one of the most robust viral clearance processes. Contemporary small virus filters reliably achieve greater than 4 logs of reduction of non-enveloped viruses in most processes, allowing greater focus on development activities related to maximizing volumetric throughput and yield. Nonetheless, the recent development of high titer spiking stocks has led to increased challenges for ensuring high virus removal while maintaining high product throughput. In order to further investigate the effect of high titer spiking stocks on viral clearance with Planova™ filters, we performed clearance studies using PP7, a small non-enveloped bacteriophage, as a model for viruses such as parvoviruses. Logarithmic reduction value (LRV) was found to be dependent on the overall challenge titer, and residual virus in the filtrate increased significantly when the viral retention capacity of the filter was exceeded. Currently, our recommendation is to perform viral clearance studies using a total virus challenge of no more than 11 log PFU/m2 to mitigate the risk of over-spiking the filter. However, for those processes with Planova filters for which higher LRV is critical, we propose using two Planova filters in series. Using this filters-in-series strategy, we were able to achieve over 5-fold increase of product throughput with complete PP7 clearance (LRV ≥ 7 ). This alternate process, with an optimized control strategy, takes full advantage of the reliability and performance of Planova filters while achieving high viral clearance that is only possible with high titer virus spiking.

R10 Chromabolt® Prepacked and Pre-Validated Columns: Three-Resin Validation Approach Santosh Rahane, EMD Millipore Pre-packed chromatography columns have demonstrated their value in ease-of use to the process development scientists, especially in pre-clinical and early clinical phase. Chromabolt® products are a family of pre-validated columns prepacked with EMD Millipore chromatography resins, designed for early clinical stage manufacturing. The Chromabolt® columns are available in 3 sizes - 10, 20 and 32 cm inner diameter (i.d.), and free up end users valuable time and resources by eliminating manual packing and cleaning. The column manufacturing process, which involves automated packing and sanitization in clean room produce ready-to-use columns with highly consistent (more than 99% of success rate) HETP, Asymmetry and flow properties. The columns' ergonomic design enables both the safety and ease of use. Additionally, carefully designed shipping method ensures that the columns arrive at end users' facilities safely and intact as proved by our shipping validation tests. The current work with Chromabolt® columns focuses on the recently launched columns that offer prepacked columns of Fractogel® COO, Fractogel® SE Hicap and Eshmuno® Q resins in addition to the current Chromabolt® columns offering prepacked Fractogel® TMAE Hicap, Fractogel® SO3 and Eshmuno® S resins. The development of the Chromabolt® columns with the new resins was achieved by adapting a three-resin approach in combination with the Taguchi model for statistical experimental design. This approach allowed us to conduct a complete validation of all the three resins at several packing conditions and in all i. d. columns. The success of standard shipping validation tests ensured column integrity after shipping. In summary, the three new resins were successfully accommodated into the Chromabolt® formats without any change in the current packing processes and with high consistency of packing method and characteristics.

R11 Improved design of Continuous Countercurrent Tangential Chromatography (CCTC) for Single-Use mAb Capture Boris Napadensky, Chromatan Chromatan's CCTC is a column-free mAb capture technology that provides a single-use continuous unit operation that overcomes many limitations of packed bed chromatography. CCTC replaces the stationary phase of a packed column with a moving slurry that is continuously pumped through several cascades of static mixers and hollow fiber membranes. This work showcases a new system design which resulted in significant simplification and scale down from the previous versions of CCTC. In collaboration with Regeneron, mAb capture and purification from a clarified CHO harvest was performed with run time of 8 hours and batch volume of 2 Liters. The system showed >90% yield while operating at low system pressures (< 10 psi). 3X improvement in HCP clearance, and 30% improvement in aggregate clearance was shown when compared with the packed protein A column

R12 Polishing of Monoclonal Antibodies using a Polymer Grafted Cation Exchanger Jonathan Royce, GE Healthcare Life Sciences The polishing steps of monoclonal antibodies present several challenges. One of the major goals of the purification process is to reduce the amount of aggregates. Typically, cation exchange chromatography is included in the purification scheme to address this problem. A new grafting technology has been developed, enabling attachment of polymeric surface extenders with cation exchange functionality onto agarose beads for use in polishing of monoclonal antibodies. The performance of these polymer grafted cation exchangers has been investigated and will be presented. The polishing step for different monoclonal antibodies was optimized regarding buffer composition and pH using 96-well filter plates and later confirmed in packed bed using column format. Yield, aggregate- and HCP-removal was monitored throughout the study. Screening conditions for binding, selectivity and optimization of elution conditions are presented, as well as comparison with CIEX chromatography media on the market.

R13 Applying QbD Based Approach to the Characterization of a Downstream Process - A Case Study Yi Jian Miao, Shire A systematic Quality by Design (QbD) approach was applied to the characterization of the downstream process for the manufacturing of a therapeutic protein product. The product critical quality attributes (CQAs) were first determined. For every downstream unit operation, a FMECA process parameter risk assessment against process performance and product quality was performed. Following risk evaluation and prioritization, the higher risk parameters were included in a multivariate high resolution DOE study to evaluate the parameter effect and potential interactions. Univariate studies were also performed for selected process parameters. The studies linked process parameters to CQAs and process performance, and generated unit operation heat map for the CQAs evaluated. Appropriate control strategy can then be developed to ensure manufacturing success.

R14 Design of a novel agarose based resin platform Hans Johansson, Purolite The first agarose based chromatography beads were introduced by Hjerten already in 1962. Fifty years later beaded agarose has become the dominant resin for protein purification and is extensively used, from research scale in sub ml volumes, to full scale manufacturing in > 500 litre chromatography columns. However, since the introduction of Sepharose 6 Fast Flow in 1982, relatively few homogenous agarose based matrices designed for large scale manufacturing, have entered the market. The focus of recent research has been more on increasing capacity and selectivity through different grafting technologies and ligand developments rather than improving the inherent properties of the agarose beads. In this paper we have used new technologies to produce beaded agarose of different particle sizes ranging from 40 - 100 µm. The objective has been to improve pressure flow properties while creating a porosity structure optimal for protein chromatography. The result indicate that there are good possibilities to design homogeneous agarose resins for large scale manufacturing, with significantly improved pressure/flow properties, capacity, and resolution compared to what is commercially available today. Application and characterization data from a set of novel agarose based ion exchangers will be presented.

R15 Continuous Downstream BioProcessing by Coupling CadenceTM Single-Pass TFF with Chromatography Steps Catherine Casey, Pall Life Sciences Pall's patented SPTFF technology is enabling continuous bioprocessing by eliminating the conventional TFF recirculation loop and achieving 2-20X concentration factors in single pass. Since SPTFF can be a key process intensification tool and operated virtually anywhere within a given bioprocess, this work explored coupling SPTFF with affinity, ion-exchange and membrane chromatography steps and evaluated the versatility and robustness of its performance. The ability of the SPTFF module to stably concentrate four different feedstocks (Fab, mAb, Bovine IgG, and alpha-amylase) by a factor of 4-5X prior to the chromatography load step was demonstrated. Further testing performed with the Fab in E.Coli periplasmic extract utilizing MustangS XT Acrodisc® indicated that the process could be operated over a broader range of operational productivity within 4-11X. Coupling SPTFF with chromatography steps not only reduces chromatography load times and/or results in noticeable materials savings, but also enables downsizing of respective downstream unit operations for significant cost savings. The smaller equipment, tank sizes, and hold-up volumes also result in smaller system footprints, which are especially beneficial for manufacturing spaces where facility fit is a concern. Indeed, reduced volumes increase the likelihood of adoption and further utilization of other single-use technologies for additional cost savings.

R16 Use of Virus-Like Particles as Live Viral Surrogates during Downstream Process Development Viral Clearance Studies David Cetlin, MockV Solutions Viral clearance is a critical downstream process attribute, however, the costs and logistics associated with live virus-spiking studies make data difficult to attain during process development and characterization. Non-Infectious virus-like particles (VLPs) that mimic the physiochemical properties of natural viruses could be utilized to generate this data. VLP's of Mouse Minute Virus (MMV) were created as "viral surrogates" and used as "spiking" agent in mock viral clearance experiments. The VLPs were produced by cloning the MMV capsid gene, VP2, into a baculovirus expression vector, expressing it in Sf9 culture and purifying the resulting particles through density centrifugation. A physiochemical comparison of the VLPs' surface charge and size was made against MMV. Quantitative ELISA assays were developed utilizing natural and recombinant VLP surface epitopes. A viral-spiking study was conducted using MMV VLP as the spiking agent. Spiked NS0 supernatant was purified through a Q-SFF column. Load and flow through pool collections were analyzed for VLP quantity via ELISA. This data was compared against a standard curve generated using a dilution series of VLP and hence, LRV was determined. The results show promise that VLPs could be employed during process development and characterization as a tool to predict viral clearance efficacy.

Miscellaneous

O1 Development and characterization of an orbitally shaken 12 L bioreactor suitable for mammalian and microcarrier cultures David Laidlaw, Kuhner Shaker Inc On the pharmaceutical market there is a demand for single-use bioreactor alternatives to rocking bags for culturing mammalian cells in the 2L-10L scales. Orbitally shaken vessels expose cells to low shear stress, making them suitable for the cultivation of shear sensitive organisms. In this work a disposable 12 L culture vessel was characterized. The vessel was constructively extended to a bioreactor with the integration of an online monitoring system (pH and DO). The influence of the integrated sensors and microcarriers on scale up parameters was investigated. This set of data presented will support all users of shaken bioreactors to optimize their specific cell cultivation conditions.

O2 The Use of Mobius MIX Vessels with Protein Solutions: Impact of Impeller Flow and Particle Shedding on Protein Turbidity and Aggregation Dana Kinzlmaier, EMD Millipore The increasing implementation of single-use equipment and assemblies in biopharm processes has resulted in more flexible facilities with faster batch-to-batch and product-to-product turnaround times. However, it has also resulted in new questions about how the materials of construction and system functionality might impact the drug products being produced. Customers considering a move to single-use mixing systems for buffer preparation have typically wanted information about particulate count and generation and extractables/leachables identity and levels as well as guidance on an effective mix protocol to ensure robust dissolution of buffer components. But when considering these same mixing systems for protein pools, additional concerns are raised about the potential impact of the impeller action and the particulates on protein quality and stability. To have a better understanding of the use of the Mobius MIX family for protein pools, we performed a study to determine whether we could detect a change in protein quality during mixing and whether the change could be correlated to a mixing parameter. In addition, we evaluated whether the particulates that are generated have an impact on short-term protein stability.

O3 Efficient separation of antibody light chains from bi-specific antibody monomer using mixed-mode sorbents Catherine Allioux, Pall Life Sciences Bi-specific antibodies are currently considered as one of the most promising class of next generation therapeutic molecules and in the coming years a growing number of such products are expected on the market. Purification of bi-specific antibodies presents unique challenges compared to that of monoclonal antibodies, and chromatographic sorbents will therefore have to answer those specific requirements. In the present study, the use of mixed-mode sorbents as capture step for the purification of a bi-specific antibody was investigated, with specific focus on the separation of the abundant antibody fragments (light chains) from the monomeric form. Three mixed-mode sorbents were evaluated for their performance in antibody light chain elimination. Our data indicate that two out of the three evaluated sorbents allowed efficient removal of antibody light chains thanks to the possibility to selectively elute antibody from the column while retaining fragments on the resin. Up to 97% pure monomers were recovered after only one purification step using mixed-mode sorbents while the initial monomer purity was about 30%. Concomitantly, satisfying recovery and efficient CHOP removal (> 1log) were obtained. Altogether, these data reveal that mixed-mode chromatography is a powerful tool to address the future challenges of purification of the growing bi-specific antibody class of biomolecules.

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

Vaccine Development & Production Summit

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