Production and Economics

Friedrich Nachtmann, head of biotech cooperations in biopharmaceuticals at Sandoz GmbH

NAFT: Npro Autoprotease Fusion Technology

Microbial expression systems play an important role in the biopharmaceutical industry. A robust, scalable, and well-understood process, reduced development times, and competitive costs are requirements for successful manufacturing. In cooperation with the Austrian Center of Biopharmaceutical Technology, Sandoz has developed a platform Escherichia coli expression technology that provides high expression levels and rapid process development for a broad range of peptides and proteins. The Npro fusion technology is highly suitable for difficult-to-express molecules (e.g., toxic peptides and proteins). Quantitative autoproteolytic cleavage at the desired and authentic N-terminus is simply induced by changing buffer conditions without costly additives or additional process enzymes. Furthermore, the fusion partner may be used for integrated bioprocessing (by combining purification and cleavage steps). NAFT represents a cost-effective technology for recombinant manufacture of peptides and proteins even at large scales.

Balazs Hunek, senior research and development manager of biopharma research and development at Praxair, Inc.

Process Advancements in Lyophilization

This poster presents two important advancements relating to commercial-scale lyophilization, the leading process for gently stabilizing sensitive products for long shelf life. The first lyophilization process improvement A) reduces drying time in the 30-40% range, B)improves product uniformity, and C) better preserves product activity. The second relates the benefits of cryogenic refrigeration of lyophilizers — using liquid and/or gas nitrogen — to mechanical refrigeration. In general, cryogenic refrigeration increases system reliability and operating flexibility by, for example, enabling rapid and constant cooling well below −50°C with high turn-down capability. Cryogenic refrigeration also lowers system maintenance, footprint, and environmental impact. Advanced high-efficiency, nonfreezing cryogenic systems offer high precision of process control and comparable cost of ownership to mechanical systems through applied engineering innovation. At the same time, contracting for reliable and cost-effective industrial gas supply is critical for cryogenic systems.

Jennifer Martinez, manufacturing technical specialist III in manufacturing sciences and technical services at Genentech, Inc.

12K Bioreactor Glucose Delivery Optimization

In an effort to improve operational efficiency of the Vacaville cell culture manufacturing plant, a new process was developed to prepare and deliver media feeds to the bioreactors. The new process used existing, idle equipment within the plant alongside several new pieces of single-use technology. The new process prepares and treats the feed media as a large batch and then fills it into single-use bioprocess containers (bags). This project significantly reduced raw material and labor costs as well as the use of constrained process equipment.

Chiali Liu, senior manager of purification operations in the pharmaceutical development pilot plant at Centocor R&D

Determining the Feasibility of Using MabSelect SuRe Resin on Multiple Products

Protein A affinity resins are used for initial capture of monoclonal antibodies, and they are among the most costly resins used in the pharmaceutical industry. Industry practice is to use a single resin batch dedicated to each specific product. After the product development cycle is complete, these resin batches are discarded. If it could be demonstrated that resin cleaning is effective, it would be feasible to use a batch of resin on multiple products. Significant cost savings in resin purchase could be realized potentially for non-GMP operations. MabSelect SuRe resin, used for initial product capture at Centocor, was tested to determine the effectiveness of cleaning procedures. The procedure for this resin requiring sodium hydroxide instead of guanidine-HCl provides a cost saving. A batch of MabSelect SuRe resin packed in a 25-cm diameter, 10-L column was used for the cleaning study. This study was performed at Centocor’s development pilot plant, which is operated in a non-GMP environment. The MabSelect SuRe column was used on five different Centocor products spanning 11 campaigns. In addition to product capture, postsanitization mock elutions were performed before and after each campaign to collect samples for assessing the cleanliness of the resin. Samples were collected throughout the campaigns and mock elutions, and assays performed included host-cell DNA, protein A ligand, endotoxin, bioburden, and protein. Cleaning effectiveness was achieved when the carry-over product quantity is lower than the maximum allowable carry-over (MAC) of product from batch to batch. Overall, the assay results indicate that this batch of resin could be cleaned sufficiently, supporting the proposal that this resin could be used on different monoclonal antibodies (MAb) at either preclinical or development stage. This outcome provides a significant operational cost savings for the development pilot plant.

Scaling Up from Bench through Commercialization

Andrew Sinclair, director of development at Biopharm Services

Developing an Effective Knowledge Management Framework for Biopharmaceuticals

Process development and manufacturing for biopharmaceuticals are often disjointed activities. Disconnects among groups can be aggravated by inconsistent terminology and poor data management. As a result, valuable information is often lost or overlooked. This is no longer acceptable given the current focus on quality by design initiatives and pressures to reduce costs and time to market. Communication between process development and manufacturing groups can be dramatically simplified if a common process knowledge management platform is used. Such a platform can streamline technology transfer over the product lifecycle, foster process understanding, and enable early views of project costs and facility fit. Adoption of the ISA-88 standard for batch control allows process and facility data to be separated and processes reapplied at different locations and scales. Key process information can then be linked to supporting data to allow assumptions and decisions to be systematically recorded. Potential also exists for process recipes to be exported to the growing number of manufacturing execution systems (MES) and control packages based on ISA-88. Ultimately the goal of the platform is to enable feedback from manufacturing performance to guide process development and create a model that supports continuous improvement and better process understanding.

Molly McGlaughlin, vice president of sales and business development at Cytovance Biologics

Successful Transfer and 200-Fold Scale-Up of a Recombinant CHO Cell Fed-Batch Process Using a New CGMP Compliant Basal and Feed Medium Combination

A research process developed in shake flasks and 0.6-L bioreactors was transferred from an independent development laboratory and rapidly scaled up to a 100-L production bioreactor. A newly developed, commercially available, chemically defined basal and feed medium combination designed specifically for CGMP fed-batch production was a key element in this success. A single 5-L bioreactor run using 10% (v/v) feeds on days 2, 5, and 7 with glucose supplementation (=1 g/L) indicated a need for a more aggressive feed schedule in order to replicate the productivity and culture properties obtained at the research scale. Without further experimentation, the feed schedule was changed to 10% (v/v) feeds on days 2, 4, 6, and 8 without glucose supplementation. In a 5-L bioreactor, the modified feed schedule replicated the 900 mg/L productivity of the research bioreactors and exhibited similar culture and metabolic properties. The modified process was incorporated easily into a formal production record for a 100-L bioreactor by the GMP manufacturing group. The feed schedule and bioreactor operating parameters transferred successfully to the 100-L bioreactor without further modification. The productivity was again 900 mg/L, and the culture properties replicated those seen during process development.

Brian Benoit, director of customer support in research and development at BioProcessors Corporation

Scale-Down Process Optimization Using the SimCell System: An Automated, High Throughput MicroBioreactor Platform

Bioprocess development requires using scale-down models at every step: clone selection, media optimization, process development, and characterization. However, these scale-down models have limitations. Bioreactor experimentation is often prohibitively expensive, and simple models such as well plates, tubes, and flasks are only roughly predictive of process outcomes in bench-top (or larger) bioreactors. Unfortunately, due to the large numbers of trials required, early stage work such as batch clone evaluation experiments typically lack the measurements and process controls present in the production bioreactors. Such experimentation using simplified processes identifies the best clone/media/process from the serial selection but cannot consider interactions between variables — and will usually miss the global optimum. The SimCell system solves this problem by providing many measurements and controls commonly found in bioreactors. It performs fed-batch protocols with pH, DO, and glucose control across hundreds of microbioreactors. Cell density, metabolites, titer, and product quality profiles permit analysis using multiple metrics. Such content provides more predictive experiments earlier in the development process. Very high throughput allows coverage of wider design spaces with greater statistical depth, providing better optimization and process robustness information. SimCell measurement and control capabilities will be demonstrated along with its use in cell culture development.

Cell Culture and Upstream Processing

Louis Cheung, engineer I in late-stage cell culture, process research and development, at Genentech, Inc.

Development of an Automated High-Throughput System for Cell Culture Process Development

Cell culture process development is currently performed at different scales of shake flasks, as well as bioreactors. The shake flask or bioreactor process generates very limited data per experiment and is time consuming. The current process is not ergonomic because it requires repetitive pipetting and sampling of the shake flasks or bioreactors. Therefore, we developed an automated high-throughput system capable of handling suspension cell cultures to improve the efficiency of cell culture process development. The Biomek FX high-throughput liquid-handling system was modified and integrated to prepare reagents and media in a 50-mL TubeSpin conical tube used as a suspension cell culture vessel. High-throughput osmolality and pH measuring technologies were developed and integrated with the Biomek FX liquid-handling system for automataic osmolality and pH adjustment during media preparation. The Quanta SC MPL automated high-throughput flow cytometer was used to monitor viable cell density, viability, and cell cycle profiles. Daily monitoring of the above-mentioned parameters was carried out to expand process knowledge. The automated high-throughput system is designed to set up and handle 384 TubeSpin tubes per experiment and deliver at least 10 times higher throughput than a conventional shake flask system. The technology also can be applied toward improvement of clone screening, media optimization, and process optimization throughputs.

Meg Tung, research associate in late-stage cell culture, process research and development, at Genetech, Inc.

Evaluation of an Integrated Analyzer for Mammalian Cell Culture Monitoring

Mammalian cell culture processes for production of biotherapeutic proteins are typically monitored through several off-line analyzers to track levels of key nutrients and metabolites. The new BioProfile FLEX system manufactured by Nova Biomedical combines the functionality of three off-line analyzers into one device, thus reducing sampling complexity and electronically consolidating data collection. In this study, FLEX was evaluated against Genentech R&D labs’ current instruments: the Nova BioProfile 400, Beckman-Coulter Vi-Cell AS, and AI Osmometer 3900 models. The evaluation includes data gathered by 20 operators at two sites using 13 different CHO cell lines. The results suggest that cell culture data collected from the FLEX can be considered equivalent to that obtained from the instruments currently used in Genentech R&D labs. Observations from each analyte measured are discussed, as well as extensive side investigations to understand differences between instruments. Key process decision parameters such as glucose concentration and pH correlated particularly well between the FLEX measurements and those of the other instruments.

Christina Bevilacquak, research associate in late-stage cell culture, process research and development, at Genentech, Inc.

Metabolism-Targeted Media Optimization Yields Significant Increase in Product Titer Across Several CHO Cell Lines

Metabolism-targeted media optimization efforts evaluated the right balance of nutrients involved in the primary CHO cell energy metabolism, such as key amino acids, monosaccharides, glycolytic intermediates, and vitamins essential to optimal cell growth and productivity. Several full factorial studies evaluating four key amino acids were performed using shake flasks and a novel high-throughput cell culture system. Studies also investigated concentrations of components aimed at reducing formation of lactate and accumulation of ammonia, detrimental waste products that can affect culture performance and glycosylation. Optimization of carbon and nitrogen ratios was assessed by improvements in cell growth and productivity. Experiments across several CHO cell lines yielded significant increases in product titer by optimizing the ratios of media components involved in CHO energy metabolism.

Erika Hanley-Onken, bioprocess product manager at Millipore Corporation

Impact of Hydrolysate Ultrafiltration on Cell Culture Performance and Filtration Characteristics

Hydrolysates (peptones) are widely used in biopharmaceutical manufacturing to enhance cellular growth and production. Ultrafiltration is an effective method for removing large-molecular-weight entities, including endotoxin from hydrolysates. However, some data suggest that ultrafiltration negatively affects hydrolysate performance in cell culture media. Moreover, the necessity of ultrafiltration before using hydrolysate in cell culture media has not been adequately established. In this case study, multiple lots of LucraTone Soy Phydrolysate were evaluated in both ultrafiltered and nonultrafiltered forms to determine their impact on cell culture performance in mammalian and microbial cultures.

Tobi Limke, bioprocess field marketing manager at Millipore Corporation

Recombinant Human Transferrin Enhances CHO Cell Growth and Protein Production

The biopharmaceutical industry is moving away from serum-containing media and animal supplements because of safety and process concerns. To minimize these risks, researchers are challenged to come up with suitable nonanimal-source supplements that provide equal or enhanced cell culture performance compared with traditional animal-derived supplements. Availability of new animal-free (AF) recombinant proteins allows for biological (not chemical) substitution for animal-derived products. CellPrime rTransferrin is a recombinant human transferrin that provides an animal-free alternative to iron salts and animalderived transferrin for industrial cell culture. Supplied as a human holotransferrin analogue, CellPrime rTransferrin binds specifically to the transferrin receptor, thereby facilitating iron uptake into the cell for maximal cell culture performance. Here, we describe cell culture data demonstrating that CellPrime rTransferrin shows equivalence to human transferrin and commonly used iron salts (and superiority to bovine transferrin) in stimulating cell growth and productivity in CHO cells.

Lloyd Waxman, chemist in analytical services at West Pharmaceutical Services

A Novel Container System for Cell Therapy Products

This study investigated the suitability of Daikyo Crystal Zenith (CZ) plastic container systems to store biopharmaceutical preparations at low temperatures (-85°C) or cryopreserved (-196°C). CZ vials (0.5, 5.0, and 30 mL) with several closure systems were filled with mesenchymal stem cell line and stored at -85°C or -196°C. Vials were tested for (a) durability and integrity utilizing a 1-m drop test, (b) viability and functionality of cryopreserved cells, and (c) thermal transfer efficiency of the material upon thaw in a 37 °C water bath. As controls, polypropylene vials were used. In the drop test, no damage or cracks were found on vial surfaces or closure systems. Dye immersion studies indicated container integrity with no failures. Storage had no effect on vial integrity after a drop test. Postthaw cell viability tested by dye exclusion was >95%. Cultures were 70% confluent within five to seven days, consistent with nonfrozen controls, and indicative of functional recovery. Doubling rates for CZ vials were statistically identical to frozen and fresh controls. The average thaw times for 0.5-, 5.0-, and 30-mL vials were four, five, and 15 minutes. Control vials exhibited a thaw time of three minutes. CZ vials are suitable for low-temperature frozen and cryopreserved storage of cells. Vials are durable and allow for preservation and maintenance of cell viability and functionality. CZ vials are optically clear, have an improved extractable profile, and offer lower permeability to gas and moisture compared with polypropylene.

Ciaran Brady, associate director of fermentation and cell culture sciences at Human Genome Sciences Inc.

Variability Observed During Scale-Up of a Recombinant Protein Production Process in S. cerevisiae and Effects on Downstream Processing

Variability in the performance of upstream processes can significantly affect the yields and performance in the downstream process. Upon scale-up of a fermentation process for production of a recombinant protein in Saccharomyces cerevisiae, subtle changes in the performance of the upstream process had a significant effect on performance and yields downstream. Specifically, changes in feed rate and pH control due to equipment upgrades had a significant effect on process performance. This presentation will discuss the nature of the process changes observed upon scale-up, the effect these changes had on the yields, and how the process was quickly and effectively adjusted to resolve the issues. The presentation will also discuss how effective controls were introduced in the process to improve process robustness and reproducibility.

Laurelle Sciola, senior product manager in the bioprocess division at Millipore Corporation

Filtration and Mixing Characteristics of Hydrolysate-Containing Cell Culture Media

Cell culture media is a complex mixture of synthetic and biological components that, in an aqueous solution, provides the proper chemical environment and necessary nutrients for healthy cell propagation and high protein expression. In the evolving technology of mammalian cell culture growth media, change is driven by the expanding variety of production cell lines and by concerns for product safety and purity. Dissolution of cell culture media, such as LucraTone Soy P supplement in DMEM basal media, can be facilitated by single-use mixing systems. Disposable mixers and filtration assemblies provide operational advantages by eliminating clean-in-place (CIP) and steam-in-place (SIP) process steps, reducing water use and decreased WFI consumption. In addition, the use of disposables can reduce the risk of microbial contamination.

Susanne Corisdeo, senior associate scientist in cell biology at Centocor

Use of Flow Cytometry to Screen and Predict Stability of Candidate Manufacturing Cell Lines

Selection of high-producing cell lines for therapeutic proteins is time consuming and labor intensive. Following transfection, selection, and expansion, a number of high-expressing clones are cultured for many generations to determine the stability of the integrated transgene. It is essential that productivity remains stable throughout culture expansion for the successful long-term production of a recombinant protein. Flow cytometry can be used to measure intracellular recombinant protein expression levels and assess the degree of heterogeneity in expression by a population of cells. Here we present several studies analyzing stably transfected cell lines for intracellular protein expression levels by flow cytometry at various points over several passages with and without selective pressure. Existing production cell lines with varying levels of stability were analyzed by flow cytometry to determine correlation between intracellular expression and product titer. In addition, flow cytometry analyses were performed during new cell-line development efforts at both the parental and subclone stages to determine their utility for predicting cell-line instability. Our findings demonstrate that flow cytometric analyses of intracellular recombinant protein expression levels from early passage cell lines in the absence of selective pressure correlate well with cell-line stability and can be used as a predictive indicator for stability of candidate manufacturing cell lines.

William Paul, senior associate scientist in research and development at Invitrogen Corporation

Replacement of Undefined Components in a Recombinant CHO Culture System While Maintaining Product Titer

Proteins, hydrolysates, and lysates of plant or yeast origin are commonly used in large-scale manufacturing processes for human biotherapeutics. Lot-to-lot variability in the composition of these constituents is well known and can affect multiple biological performance indicators. We sought to remove all undefined components from a hydrolysate-containing process while maintaining product titer. The cells used for this study were from a recombinant CHO cell line engineered with a CHEF1 expression vector to express IgG4. These cells were originally grown in medium supplemented with both protein and hydrolysate. Product titer had been observed to vary up to twofold as different lots of hydrolysate were used. The cells were first sequentially adapted from the undefined, hydrolysate-containing medium to a chemically defined medium. Then, using DOE methods, we identified two chemically defined basal formulations that achieved comparable peak viable cell density but lower productivity when compared with the original medium. Results of spent media analysis were used to develop a complementary feed strategy, which was then tested in bench-scale stirred tank bioreactors. The resulting chemically defined medium and feed supplement supported peak viable cell density and IgG expression, exceeding that of the undefined fed-batch control culture. This work was sponsored by ICOS under a media development agreement between ICOS and Invitrogen.

Jenkuei Liu, senior staff scientist in the applied market department at Applied Biosystems

A Robust Sample Preparation and High-Sensitive Assay for Detection of Residual CHO DNA

Accurate quantification of residual CHO DNA is essential for biopharmaceuticals produced by CHO cell lines. We have developed a high-sensitive CHO DNA detection process that can quantitate <1 pg of DNA in 1 mL of six samples from sample preparation to results in four hours. The sample preparation procedure uses magnetic particles that recover >85% of 0.1 pg CHO DNA spiked into diverse solutions (having extreme pH values or high concentrations of salts) typically used in purification of proteins by chromatography. The same recovery efficiency was also demonstrated in samples with protein of 150 mg/mL. This procedure can also be automated to simultaneously process multiple samples. The newly developed CHO DNA quantification assay uses real-time PCR and a new PCR reagent that tolerates high concentrations of inhibitors. This assay has high PCR efficiency and shows a wide dynamic linear amplification across at least seven logs of concentrations. It allows accurate quantification down to 3 fg of CHO DNA in the PCR reaction. Detection sensitivity and PCR efficiency are not affected by severe degradation of CHO DNA. The assay detects DNA from different CHO cell lines with similar sensitivity and is highly specific to CHO DNA.

Leigh Pierce, president of Pacific GMP

Versatility of a Single-Use Bioreactor Platform for Culture of Diverse Cell Types

Commitment to a single cell-culture platform for facilities that require culture of different types of cells can significantly reduce equipment costs and minimize the risk of cross-contamination as well as the need for cleaning, sterilization, and validation. An important consideration in the choice of a single-use system is the ability of the platform to support growth and productivity of various cell types. Configuration of the cell culture system will affect the shear forces experienced by cells as well as the mass transfer of gases. Different types of cells have different sensitivities to such parameters. Generally, mammalian cells are more sensitive to shear forces than bacteria, but bacteria are more sensitive to oxygen limitation. The bioreactor platform used in these studies consists of a presterilized, flexible, disposable, plastic bag placed on a rocking platform and filled with media and a gas mixture. Growth of Escherichia coli, CHO, and hybridoma cells are each supported in these bioreactors as measured by the daily cell density, the consumption of nutrients and production of metabolites. These data demonstrate that this platform is flexible enough to support growth of vastly different types of cells.

Stephen Gorfien, director of research and development at Invitrogen Corporation

Novel Approach to Chemically Defined Platform Medium and Feed Optimization

Developing fed-batch media platforms with chemically defined base medium and feed for bioproduction cell lines is traditionally a difficult and time-consuming process. Historically, strategies used have ranged from the simple iterative process of cell-based assays and spent media analysis to highly complex DOE designs also requiring multiple iterations of cell-based assays followed by data analysis. This methodology typically results in optimization that is highly clone specific. It has been challenging to translate this methodology and its results into a platform optimization system. Using multiple approaches, we have identified a way to develop well balanced, chemically defined cell culture platforms consisting of base medium and concentrated feed formulation with sufficient diversity to support fed-batch optimization. This approach results in optimized base medium, feed formulation and fed-batch process for both the high-density growth and the recombinant protein production for many recombinant CHO cell lines expressing different proteins including IgG from different parental CHO cells.

Qiang Li, chief scientfic officer at ATGCell Inc.

REEMAC: A Solution for Recombinant Protein Production and Stable Cell Line Establishment

We have developed an exogenous expression system called REEMAC to quickly produce recombinant proteins from cultured mammalian cells. To achieve higher expression levels for recombinant proteins, we created a set of new vectors by examining a variety of expression elements and optimizing the entire expression cassette. We demonstrated that expression of proteins can be dramatically enhanced through use of proper combinations of different elements. Furthermore, we also optimized cell culture conditions. We have applied REEMAC to express >100 target proteins including cytokines, growth factors, neurotrophins, surface antigens, receptors, transcription factors, kinases, and proteases. The candidate cell lines can be generated within five to six weeks. In HEK and CHO cells, 50-120 pg/cell/day of recombinant cytokines was achieved. Also, REEMAC can also be used to efficiently generate difficult-to-express stable cell lines for pharmaceutically important targets, such as G-protein coupled receptors, nuclear receptors, ion channels, and transporters. We have generated >30 stable cell lines expressing target proteins. Furthermore, we also developed ExpressBoost reagents for further boosting exogenous protein expression in mammalian cells.

Sally Grosvenor, scientific communications manager in applied research and development at Novozymes

Advances in Animal Free Media Supplements for Optimal Cell Culture Performance

The biopharmaceutical industry has seen a shift away from the use of serum in upstream manufacturing processes primarily driven by regulatory concerns over biological contaminates derived from serum. The ideal media for today is robust, containing defined, animal-free supplements that deliver optimal cell growth, productivity, and consistency. In this study, we examine recombinant forms of three key serum proteins, transferrin, IGFI, and albumin, and report on their ability to stimulate cell growth and productivity. CellPrime rTransferrin is a recombinant analogue of human transferrin that has been shown to be equivalent in potency to human transferrin and more potent than other iron supplements. The industry-proven LONG^®R3IGF-I growth factor supplement is manufactured specifically for the cell culture market and has been shown to be equivalent to or better than insulin in stimulating mammalian cell growth and productivity under serum-free conditions. We also demonstrate that the two in combination achieve synergistic performance with respect to cell growth and productivity. CellPrime rAlbumin AF-G animal-free albumin is designed specifically for large-scale industrial cell culture. This study examines four pilot batches of the CellPrime rAlbumin AF-G supplement and compares its effect in serum free medium with commercially available BSA.

Jouni Ahtinen, Explorer product manager at Medicel Oy

Role of the Known Transcription Factors of Yeast Saccharomyces Cereviseiae Investigated with a High-Throughput Cultivation Platform

Our objective was to acquire new information of the effects of certain transcription factors of the yeast Saccharomyces cerevisiae in anaerobic conditions. Measured variables were growth rate, yield of biomass from glucose, extracellular ethanol, glycerol, and acetate. The study was conducted using transcription factor deletion strains and comparing the measurement results with corresponding results obtained from a wild-type yeast strain. We investigated 52 different deletion strains with the Medicel Explorer equipment and carried out 120 individual batch cultivations within a 10-week period. More than 2,500 quantitative metabolite analyses were performed in real time using the Medicel Inline HPLC during this project. One laboratory technician executed the entire project. Statistical differences in yields and growth rates were studied with at-test. Twelve yeast knock-out strains differed statistically from the wild-type yeast strain by their yield of ethanol, 12 by their yield of glycerol, three by their yield of acetate, 11 by their yield of biomass, and 10 by their growth rate. Application of principal component analysis (PCA) showed that ethanol and glycerol yields had parallel effects on the results. Biomass yield and the growth rate also seemed to have a parallel effect.

G. Thomas John, director of marketing and innovation at PreSens Precision Sensing GmbH

New Insights in Cell Cultivation: Online Oxygen and pH Monitoring in Multidishes

Optimizing cell culture techniques is gaining significance in regenerative medicine. Two key parameters for cell activity and proliferation are dissolved oxygen (DO) and pH. With complete documentation of these parameters during cultivation, the quality of the cultures can be estimated and culture conditions or assays optimized. Using the SDR SensorDish reader, DO and pH in 24-well multidishes were monitored continuously and nondestructively. The pH or oxygen sensors are located at the bottom of each well and their signals detect noninvasively through the bottom of the multidish by the 24-channel reader. Cultivated directly in SensorDishes, the cells are not influenced by the sensor. The small and robust readers can be put in an incubator or on a shaker for constant culture conditions. Possible applications: Stem cells are cultivated under hypoxia and DO within the samples while cultivation is monitored. So far, unconsidered effects of opening the incubator or media change on the oxygen content are discovered. Furthermore, the SDR is used for online pH and DO monitoring in tissue engineering with three-dimensional cultures of chondrocytes. New insights on the cell activity at high concentration are found. The reader enables convenient screening of 24 samples in parallel for systematic process optimization.

John Carvell, director of reasearch and development at Aber Instruments

Monitoring Additional Parameters in Cell Culture Using Scanning Radiofrequency Impedance Spectroscopy

Radio-frequency impedance (RFI) is widely used for measuring cell concentrations during mammalian cell culture. Measurement is based on the principle that the permittivity of a cell suspension at low radio frequencies is raised by the presence of cells. This permittivity falls with an increase in the frequency of applied field in the radio-frequency range due to a Maxwell-Wagner type interfacial polarization relaxation process (the so-called beta dispersion). The size of this fall is proportional to the volume fraction of intact cells. Additional information about the cell suspension can be obtained from the beta dispersion if whole frequency scans are obtained rather than single-frequency measurements. In the work presented, the Cole-Cole equation was fitted to frequency scan data obtained online from cell cultures, giving not only the fall in the permittivity (De), but also the characteristic frequency and the Cole-Cole alpha as additional parameters. The fitting parameters obtained were then used not only to calculate the volume fraction of viable cells (P), but also to provide a further term rCm, which simultaneously provides information about the cell size (r) and the membrane capacitance (Cm). Although it is difficult to measure r and Cm separately, the actual value of rCm could provide additional information about the physiological state of the cells during fermentation. In particular, it can be shown that the value of rCm could be a useful tool for picking up changes in the cell membrane capacitance as cells undergo apoptosis. Because the additional parameters picked up by frequency scanning can give important information about the physiological state of cells in culture — which in turn defines cell growth, cell viability, metabolism, and productivity — multifrequency scanning can provide a useful tool in process development, optimization, control, and PAT in cell culture.

Fouque Nicolas, biotech engineer in the bioprocessing group at NovImmune SA

Accelerated Expansion of Upstream Processing Capabilities within a MAb Manufacturing Process through the Combined Establishment of a Dual CHO Cell Line Development Strategy and Disposable Technologies

Due to both expanding preclinical development and intensifying in vivo research activities at NovImmune, an accelerated and robust improvement of MAb production capabilities was a prerequisite to sustaining the rapid increase of high-quality product demands. Coupled with the GS system (Lonza Biologics), the CHOK1SV cell line was used to generate, on one hand, semistable pools for fast supply of MAb product for early preclinical development and research activities, and on the other hand, stable high-yield cell lines for late preclinical development by high-throughput screening using ClonePixFL technology (Genetix). In terms of upstream process development, the single-use Wave bag bioreactor (GE Healthcare) was operated in proprietary fed-batch mode. Furthermore, the development of primary recovery steps involving disposable depth filtration and concentration (Sartorius) was allied with disposable fluid transfer and storage. As a case study with the NI-0701 preclinical project, the dual CHO cell line development strategy led to the identification (in seven weeks) of a semistable pool with a productivity of 0.21 g/L and the isolation of a high-yield stable cell line (in four months) expressing the NI-0701 MAb at 1.3 g/L in shaken fed-batch cultures. This translated to the production of 2.5 g in 10 L and 45 g in 25 L using Wave bag fermentation development and scale-up. After robust and fast clarification and concentration steps, an overall MAb recovery of 97% was achieved, while satisfying product quality and safety by the use of disposable and consistent animal-free wetted contact surface materials. The combination of a dual CHO cell line development strategy and the application of disposable technologies ensured the implementation in less than six months of a high-performance, generic upstream process platform involving limited human resources and reducing capital and operating expenses while guaranteeing high-quality and safe product.

Mark Timmins, senior development engineer in product development at Polestar Technologies, Inc.

Characterizing Optical Oxygen Sensors Integrally Equipped into Disposable Culture Bags

Single-use rocker-style cell culture bags were equipped with optical sensors for O2. Gamma-stable patches of the oxygen sensor film were heat-sealed onto custom “optical ports” before assembly of the ports into the bags. The oxygen sensor operates on the principle of fluorescence quenching, whereby predictable variations in fluorescence lifetime were used to compute dissolved oxygen concentration within the bag. Bag sensors were read from the bag exterior by connecting to an existing optical-based process monitor. Measurement of oxygen levels in such bags was demonstrated by both chemical and biological challenges, in both the aqueous and gas phases. Measured values spanned the range of 0-100% air saturation, showed rapid response times, and excellent correlation to standard offline measurements. Once characterized, the integral oxygen sensors were used to characterize oxygen mass transfer within the bags as a function of various operating parameters (fill, rocking rate, and so on).

Changlin Dou, group leader in cell line development for PD Direct at Invitrogen Corporation

Benefits and Limitations of ClonePixFL Technology in Stable Cell Line Development

Clone selection is one of several key steps in a successful, stable cell-line development process. To overcome the shortcomings of traditional manual cloning and screening processes, recent advances in clone generation have focused on developing high-throughput clone selection technologies, including the ClonePixFL system. Based on first-hand laboratory experience, this presentation will highlight some key benefits as well as limitations of the ClonePixFL technology in applications for clonal cell line development. Case studies showcasing multiple host cell lines and different classes of proteins with design-of-experiment (DOE) approaches for rapid identification of a suitable cloning medium will be discussed. Data on development of alternate clone selection algorithms for the ClonePixFL system will also be shared. Our experience indicates that this technology has clear labor-saving advantages compared with traditional cloning methods, as long as sufficient development time and effort has been invested.

Laetitia Malphettes, engineer II in early stage cell culture at Genentech, Inc.

Development of an Alternative Vector Configuration for the Creation of Highly Productive CHO Cell Lines

Improvements to the expression levels of recombinant proteins in production cultures are highly desirable for supporting increasing demands for therapeutic protein production and can positively affect bioreactor plant capacity requirements and cost of goods. One critical component in enginering highly productive mammalian production lines is the cell-line development vector used, which determines both the selection stringency and the expression level of the desired recombinant protein. We have created a novel stable cell-line development plasmid using an IRES-driven DHFR resistance marker to functionally link expression of the recombinant protein of interest to an amplifiable selection marker. To maximize selection stringency, we engineered the DHFR resistance gene to minimize functional DHFR expression using a combination of low-use codons and a degradation domain. We combined this modified DHFR selection marker with a newly identified promoter that is significantly more potent in driving the expression of recombinant proteins in CHO cells than the widely used CMV promoter, as assayed through transient transfection. We have determined the optimal conditions for use of this new vector and will discuss its use in CHO stable cell-line development. Data will be presented on the use of this new vector showing that it is capable of driving high levels of expression of both mRNA and recombinant protein expression, with equivalent product quality to current approaches.

Ashley Smith, research and development scientist in cell sciences and development at SAFC Biosciences

Use of Chemically Defined Supplements in Optimizing the Performance of CHO Cells

Chinese hamster ovary (CHO) cells are widely used by the biopharmaceutical industry for production of therapeutic recombinant proteins. Enhancement of cellular productivity can be achieved through the use of many techniques including the delivery of nutritional supplements (feeds) during cell culture. Historically, undefined supplements have been used to increase both the cell growth and productivity in fed-batch processes. However, both regulatory concerns and variability associated with undefined components have discouraged the use of such additives, creating a need for chemically defined (CD) feeds. In response to this need, we have developed a platform of CD feeds for use with multiple CHO cell lines cultured in a broad range of media. These feeds have been optimized based on spent-media analysis to meet the nutritional and physiological requirements of CHO cells in suspension culture. Studies will be shown in which feeding with these chemically defined supplements significantly enhances growth and productivity of CHO cells, similar to the effects observed in cultures fed with undefined supplements.

Barry Rosenblatt, director of innovation in biopharmaceutical services at Charles River Laboratories

Detection of Mycoplasma/Acholeplasma/Spiroplasma Contamination in Biological Samples by Amplification of Specific Nucleic Acids and Fluorescent Probe Technology

Contamination by mollicutes is a problem associated with cell cultures, not easily detected because it usually does not produce turbidity or cytopathic effects. Their size and lack of rigid cell wall allow these organisms to pass through most sterilization filters. Traditional testing for mycoplasma/acholeplasma/spiroplasma contamination in any biological material involves culture methods that require lengthy incubation periods to detect fastidious, slowgrowing organisms. Nucleic-acid amplification techniques (NAT) offer a rapid alternative to the culture methods. A NAT-based detection assay has been developed for a wide range of mollicutes including mycoplasma, acholeplasma, and spiroplasma using RT-PCR with sequence-specific primers and fluorescent probes developed specifically for this assay. Because RNA is more labile than DNA, the extraction and targeting of RNA should decrease the detection of nonviable organisms as well as persistent residual DNA. This assay was developed based on the 2.6.7 guidelines of the European Pharmacopoeia, demonstrating a 10-CFU/mL detection limit in several matrices for A. laidlawii, M. fermentans, M. hyorhinis, M. orale, M. pneumoniae, M. arginini, M. gallisepticum, and M. synoviae, an optimal selection in terms of contaminant frequency and phylogenetic relationships. Specificity was demonstrated using species with close phylogenetic relationships to mollicutes.

Paula Ravnikar, group leader of PD-Direct at Invitrogen

Improved Cell Line Performance in Fed-Batch with High-Osmolarity-Resistant CHO Cells Obtained with Revolution Treatment

A novel approach to optimizing protein production is to engineer host cell lines to tolerate critical environmental changes. One example is tolerance to the high osmolarity of culture medium that occurs after concentrated nutrient feeding for high-cell-density cultures. To isolate cell lines with this desired characteristic, Invitrogen’s Revolution technology was applied to CHO DG44 cells to inhibit mismatch repair, thereby generating a genetically diverse cell population. After treatment, evolved cells were shifted directly into experimental conditions as high as 600 mOsm/kg. Revolution-treated cells immediately demonstrated superior growth characteristics in high-osmolarity medium without lag or additional growth adaptation. This Rev-O cell line (Revolution-treated, high osmolarity resistant) grew to higher cell densities with faster growth rates than did untreated cells. This phenotype has been demonstrated to be stable for up to 75 generations. Additional studies show that the Rev-O cell line can be transfected and methotrexate amplified to express a recombinant monoclonal antibody. Fed-batch shake-flask experiments with concentrated feeds show that Rev-O/rIgG achieved higher cell densities and higher titers than the parental DG44/rIgG. These data indicates that Revolution technology will be valuable toward systematically engineering a mammalian host cell with novel phenotypic traits that are advantageous for bioproduction.

Stefan Schlatter, associate director of cell culture technology in process science upstream development at Boehringer Ingelheim Pharma GmbH & Co. KG

Fast Track, High Expression: Fit for Commercial Scale

Boehringer Ingelheim’s high-expression system (BI-HEX) enables fast-track development of high-quality, high-titer processes for producing biopharmaceuticals from CHO cells, shortening the time to clinic. BI-HEX combines several state-of-the-art technology platforms in one concept that meets demands for shorter development times to first use of a new biologic entity in humans, and it follows the paradigm to “do it right the first time.” Early focus on commercial-scale potential, process performance, and product quality, safety, and comparability prevents unnecessary costs and delays later on. Specific productivities >50 pg/cell/day for monoclonal antibodies have been achieved and successfully translated into product titres of >6 g/L in a fed-batch process without compromising product quality, activity, and characteristics.

Recovery and Purification

Jeff Allen, research leader in analytical biochemistry for biopharmaceutical services at The Dow Chemical Company (Dowpharma)

Biopharmaceutical and Antibody Derivative Production Using the Pseudomonas-based Pfenex Expression Technology

The Pseudomonas-based Pfenex expression technology has proven to be a robust and cost-effective platform for production of numerous classes of therapeutic proteins, including various types of antibody derivatives and “scaffold” proteins. High-throughput methods allow dozens of different expression strategies and host-cell combinations to be rapidly tested in parallel. Efficient periplasmic secretion of proteins allows the formation of disulfide bonds, critical to the production of active antibody derivatives, and also the development of simplified downstream processes. Here we have purified and analytically characterized several types of antibody derivatives including a fully intact monoclonal antibody and demonstrate the versatility of our platform for expressing this important class of therapeutic proteins.

Phil Kuhlman, senior research scientist in biochemistry at SemBioSys Genetics Inc.

The Rapid Production of Lys-C from an Animal-Free Source

The growth media for the organism Lysobacter enzymogenes was manipulated to improve the secreted amount of Lys-C activity. L. enzymogenes was grown in a fermentor and clarified by centrifugation, yielding culture supernatant that exhibited a protease activity equivalent to 3 mg of Lys-C per liter of culture. A series of four different chromatography columns was used to separate Lys-C from contaminants in the culture supernatant, giving a pure preparation that exhibited the specific cleavage activity expected for Lys-C and cross-reacting with antibodies raised against a Lys-C peptide. By loading the eluted enzyme fractions from one column directly (or with minimal manipulation) onto the next column in the purification series, a rapid purification could be completed in less than eight hours. The speed of this approach enabled production of highly pure enzyme with greatly reduced self-digestion leading to higher yield. The resulting enzyme preparation was low in endotoxin, had specific proteolytic activity, and showed good storage properties.

Tony Brazzale, eastern United States regional manager in sales for BIA Separations Inc.

An Industrial Platform for Purifying Influenza Virus Particles

Avir Green Hills Biotechnology is developing innovative seasonal and pandemic influenza vaccines based on the deletion of the NS1 gene (delNS1 vaccine). The vaccine is replication defective and applied intranasally. Currently, an H1N1 monovalent vaccine is being tested in a clinical phase I study, with an H5N1 avian influenza vaccine soon to be initiated. A production and purification process will be presented that was successfully used for the pilot-scale production of H1N1 and H5N1 influenza A vaccine virus. Data will also be discussed on the selection of chromatographic media, which is relevant to eliminating downstream purification bottlenecks. Details will be given on obtained virus yields as well as impurity removal. The vaccine virus is produced in static cell culture using Vero (African Green monkey kidney) cells. After clarification the vaccine virus bulk is purified using the same scheme for all different subtypes: concentration by tangential ultrafiltration, AEX chromatography using a CIM QA monolith, and an SEC polishing step allowing for buffer exchange. This purification scheme guarantees thorough depletion of host-cell DNA and total protein. In addition, an HPLC method will be presented for quantifying influenza virus in the vaccine with the use of CIM monolithic analytical columns, and the results will be compared with the haemagglutination method.

Mark Perreault, director of biopharma membrane research and development at TangenX-NOVASEP

Developing an Economic Model for Single-Use TFF Cassettes

Developing an economic model for downstream purification process steps is a critical task that is important when scaling a process from benchtop to process scale. Preparing an accurate economic model must include all critical aspects of the process. Considerations of labor, utilities, and waste contribute to the complexity of an accurate process model. It is here that the benefits of single-use technologies must be weighed against traditional reusable process steps. Novasep (through TangenX Technology Corp.) offers the first presanitized, purposely built, single-use, tangential-flow filtration cassette for the biopharmaceutical industry.

Jon Fisher, technical manager for healthcare at Rohm and Haas Company

Use of a Polymeric Reversed-Phase Resin for Hydrophobic Interaction Chromatography

Unlike reversed-phase chromatography, hydrophobic-interaction chromatography is a technique that allows protein purification using aqueous mobile phases. Under appropriate conditions, a less hydrophobic reversed-phase chromatographic media (e.g., Amberchrom CG71) can be used as a hydrophobicinteraction resin. In this study, we demonstrate the use of Amberchrom CG71M acrylic 75-µm resin for protein purification under hydrophobic-interaction conditions. Hydrophilic proteins can be successfully separated on this resin without using denaturing solvents.

Nathalie Fraud, applications specialist in purification technologies at Sartorius Stedim Biotech

Hydrophobic-Interaction Membrane Chromatography

Membrane chromatography offers a cost-effective alternative to traditional packed-bed chromatography in flow-through operations, such as polishing for the removal of virus and contaminants in antibody manufacturing. As use of flow-through applications expands, others use the membrane chromatography for capturing large molecules. Such bind-and-elute applications imply the demand for higher capacity and larger surface membrane areas as compared with flow-through applications. With these considerations, a novel Sartobind HIC membrane was developed. The macroporous membrane structure with 1- to 3-µm pores was designed for high flow rates and binding capacities. Using the advantages of membrane chromatography with virtually no diffusion limitation, shorter processing times attributable to convective flow and ready-to-use capsule design, the hydrophobic-interaction membrane chromatography offers a new scalable and disposable tool for purification and separation of biomolecules as well as for polishing applications in the biopharmaceutical industry.

Lisa Connell-Crowley, senior scientist in purification process development at Amgen

Reducing Viral Safety Risk Through Purification Process and Viral Study Design

Although purification platforms can ensure viral safety and enable the use of generic viral validation, some processes deviate from the platform because of the molecule’s characteristics or commercial development needs. How do you ensure viral safety for an off-platform process without costly extra studies? We have developed a strategy in which process design is influenced by knowledge of operating spaces for viral clearance by various unit operations. Subsequent viral studies are designed to maximize clearance numbers when needed. This approach has been successful for recent nonplatform processes.

Jon Fisher, technical manager for healthcare at Rohm and Haas Company

Upstream Decolorization of Recombinant Products

One of the biggest challenges in biopharmaceutical production is the reduction of costs. Increased production titers in products such as monoclonal antibodies have now created bottlenecks in downstream processing. High-titer (>60 OD) bacterial cultures can be very darkly colored, and this color can bind tightly to anion-exchange media. For larger capture columns, the installed cost of a packed column can be hundreds of thousands of dollars. Therefore, it is important to ensure that the media lifetime is sufficient to provide good process economics. One strategy for improving chromatographic column lifetime is the use of inexpensive, large-particle-size anion-exchange resins for the removal of column-fouling color bodies before further purification. Amberlite FPA resins are economical choices for upstream color and contaminant removal. These resins function essentially as a guard column for the more expensive resins and reduce the need for more frequent column repacking and media replacement. In this study, we demonstrate protocols for effective decolorization of fermented product process streams. This includes protocols for both smaller molecules, such as vancomycin, and larger recombinant proteins. Results demonstrate that Amberlite FPA resins are effective for decolorizing these feedstreams while providing excellent product recovery.

Hui Liu, scientist at Oceanside PR&D for Genentech, Inc.

Development of an Anion-Exchange Chromatographic Step in Bind-Elute Mode

Anion-exchange chromatography has been widely used as a polish step in flow-through mode to remove process related impurities such as DNA, HCP, and virus particles in monoclonal antibody purification processes. Some experts argue that resin capacity and processing time are instrumental in the decision to run an anion-exchange step in flow-through mode because these are economic advantages that cannot be achieved by performing an anion-exchange step in bind-elute mode. We have shown that running an anion-exchange chromatographic step in bind-elute mode has the ability to remove not only process related impurities, but product related impurities as well. Therefore, the true benefit of performing the anion-exchange step in bind-elute mode is the ability to reduce the overall number of unit operations in a purification process. In this poster, we demonstrate how to rapidly develop a bind-elute anion-exchange chromatography step by performing only a few screening runs. The viral clearance, process-related impurity removal, and product-related impurity removal using anion-exchange chromatography in the bind-elute mode will also be presented for several monoclonal antibodies.

Tetsuyuki Saika, vice president of fine chemicals at DAISO Fine Chem USA, Inc.

Novel Polymeric Coated Reversed-Phased Silica Gel Regenerated By a 0.1 N Sodium Hydroxide Solution

Polymeric coated reversed-phase shows excellent durability against alkaline conditions. C8 polymeric-coated silica gel shows sufficient separation after 400-column-volume washing with a 30/70 0.1 N sodium hydroxide/methanol mixture. The phase is modified on rigid silica gel by in situ polymerization of C8 monomer and then end-capped using a new catalyst. Insulin separation using C8 polymeric coated phase has been investigated.

Julie Pearlman, application scientist in research and development at PhyNexus Inc.

Optimization of Protein Purification Using Small-Scale Separation Columns

Characterization of therapeutic candidates requires that proteins are well purified and enriched post expression. The process for adequate preparation requires that sufficient quantities of material be scaled up and processed using time-consuming and expensive chromatography equipment. As developments in functional and analytical assays increase throughput and reduce the amount of protein required for analysis, efficient small-volume protein purification would provide high-value information to researchers in earlier stages of drug discovery and development. Recent advances in the area of miniaturized high-throughput tools for purification, enrichment, and desalting of proteins eliminate bottlenecks associated with traditional protein purification techniques. By performing high-performance functional protein separations on small samples in parallel, it is now possible to obtain more relevant data in a completely automated format. Investigation of protein separations in small-scale extraction columns is presented along with optimized conditions enabling functional and analytical characterization of therapeutic proteins purified by this unique format.