In recent years, there has been

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1 From Discovery to the Clinic A Robust Strategy for Rapid Development of Clinical Manufacturing Processes Michael J. Gramer,, Mark D. Hirschel, and Jae Sly In recent years, there has been a rapid increase in the number of promising new biological therapeutic candidates due both to their acknowledged effectiveness and to the accelerated discovery processes associated with proteomics research. The majority of those candidates are being discovered in academic laboratories or at small biotechnology companies, which often lack the resources or know-how to move past the production of researchgrade materials. This article describes how we have used hollow-fiber technology as a robust method for rapidly providing such discovery organizations with efficient clinical manufacturing processes suitable for phase 1 production and beyond. Biovest International provides full service process development and clinical contract manufacturing of PRODUCT FOCUS: BIOPHARMACEUTICALS PROCESS FOCUS: PROCESS DEVELOPMENT WHO SHOULD READ: PROCESS DEVELOPERS, PROJECT MANAGERS, MANUFACTURING GROUPS KEYWORDS: HOLLOW-FIBER BIOREACTORS, PROCESS DEVELOPMENT, SCALE-UP, CLINICAL MANUFACTURING LEVEL: INTERMEDIATE 62 BioProcess International MAY 2003 mammalian cell products. In addition, the company manufactures hollow-fiber bioreactor instrumentation ranging from research to large pharmaceutical scale. In the typical scenario, an investigator constructs a cell line capable of producing several milligrams of researchgrade product using basic laboratory equipment. If that product shows promise, the next step is to produce gram quantities for preclinical testing and purification process development, followed by cgmp production of tens to hundreds of grams for completion of a phase 1 clinical study. The primary goal of a discovery organization is usually to complete that phase 1 study quickly and use the resulting data to attract more substantial project funding. Unfortunately, in their haste to complete a phase 1 study, discovery organizations typically undervalue the usefulness of process development during those early stages. The project design described below outlines one approach to satisfying the timeline goals of a discovery organization while still providing substantial process improvement before phase 1 production. PROJECT DESIGN OVERVIEW Figure 1 provides a complete project timeline. The requirements, goals, and priorities of each discovery organization vary considerably, as does the amount of work already accomplished by each organization. Technology transfer from the organization to us is our first step in process development. Noninvasive NMR cross-sectional image of viable cell density after 13 days of growth in a hollow fiber bioreactor. The color scale ranges from black (no cells) to green, yellow, and then red (highest cell density). The hollow fibers (black circles) have an outside diameter of 220 microns. BIOVEST INTERNATIONAL, INC. ( AND THE CENTER FOR INTERDISCIPLINARY APPLICATIONS IN MAGNETIC RESONANCE ( As a launching point to discussion, the discovery organization is asked to complete a project questionnaire. Invariably, further questions arise from information included in this document, and a follow-up conference call is required. After these discussions are complete, a project proposal is provided with a number of options, primarily related to how much process development is desired. We then work with the discovery organization to finalize a project design. The initial step in process development is to choose a production

2 Figure 1: Steps and time required to go from the bench to phase 1 clinical GMP production medium. Once that medium is chosen, preparation of a master cell bank (MCB) can begin. In parallel, the remaining process development steps continue. During this time, close communication is necessary between the process development and production groups. Technology transfer between them happens in real time through informal and formal communication tools. After completion of MCB testing, cells are thawed, scaled up, and inoculated into a bioreactor. Production continues until sufficient product (secreted protein) is recovered, after which an end of production (EOP) cell bank is created and the product is purified. On project completion, some time is needed for review of batch records for final product release. The entire process can be complete in about 10 months. For example, one client recently provided us a recombinant CHO cell line that secreted 12 µg/ml in a T-flask, and 90 g of clinical material was provided to them eight months later. PROCESS DEVELOPMENT Increased process development work leads to lower costs at the clinical production scale. Perhaps more important, such efforts result in more accurate predictions, providing a higher confidence that sufficient materials can be produced on time and within budget. However, process development efforts also have associated costs and time delays, and their return on investment can be uncertain. As a Table 1: Example media screen Complete T-flask HF Complete Estimated Medium Performance Medium Use Media Cost RPMI+10% FBS + Both Sides $140,000 Proprietary +10% FBS ++ Cell Side Only $ 19,000 CD-ACF ++ Both Sides $ 39,000 result, much initial discussion with our clients is centered on determining what level of process development is right for their needs. Cells: The cell lines we most often see are CHO, hybridoma, and NS0 cells, the majority of which are secreting a monoclonal antibody or derivative thereof. Of all the options for process development, cell line optimization offers the best opportunity for order of magnitude improvements by enhancing cell line stability, production rate, and growth characteristics. However, such studies involve either re-engineering the cell line or intensive screening efforts. As a result, cell line optimization is usually not a priority at this stage for discovery organizations because of the associated costs and time necessary to complete those enhancements. One issue that should be considered by a discovery organization when initially constructing a cell line is cost of the selection agent. At small scales, the cost of those agents is not a concern, but they can add thousands of dollars to the cost of clinical scale production. One approach is to construct cell lines that do not require expensive selection agents. A second approach is to demonstrate stability in the absence of a selection agent by repeated, controlled passage over several months, with intermittent determination of cell expansion rates and product secretion levels to support stability analyses. Bioreactor Type: A second parameter for process development is choice of the bioreactor production system. For large-scale production of secreted proteins, we use our company s ACUSYST-MAXIMIZER and ACUSYST-XCell hollow-fiber systems (Figure 2). These hollow-fiber systems are ideally suited for serving the needs of discovery organizations for a number of reasons. The systems use a technology called cycling, which is useful for reducing cell side gradients for increased and prolonged productivity (1, 2). Cycling is essential for scale-up homogeneity. The potential MAY 2003 BioProcess International 63

3 Figure 2: Hollow-fiber bioreactor schematic and typical support for a 160-mL bioreactor. Cells are placed outside the fibers of the hollow-fiber bioreactor. Medium is circulated at 0.5 L/min from a reservoir through the pump, a gas-exchange cartridge, and the inside of the fibers, and returns to the reservoir; the high circulation rate is primarily for oxygenation. Most cell nutrition comes across the fibers from the non cell side feed of about 10 L/day. The cell side feed of about 0.1 L/day is used to supply high molecular weight growth factors, and the product is harvested from the cell side at the same rate. The 10-L feed will result in about the same production as found from 10-L of medium in a tank, but the product is harvested in a concentrated format. In many cases, the non cell side medium is a simple, inexpensive basal medium that reduces media costs. Production corridor in a GMP clean room housing 12 XCell hollow-fiber bioreactor systems (16,000 L equivalent tank capacity) in 1,500 ft 2. BIOVEST INTERNATIONAL, INC. ( 64 BioProcess International MAY 2003 for crossover contamination, an important consideration for multiproduct facilities such as ours, is minimized by the fact that all the wetted components of these hollowfiber systems are disposable. Another advantage is that these hollow-fiber systems are linearly scalable: Pilot-scale production in the Maximizer is directly scaleable to production in an XCell. The semipermeable fiber membrane in the bioreactor design retains protein, and that can substantially reduce the need for expensive growth factors in the medium. In addition, the product is retained on the cell side of the fibers, resulting in a concentrated product and a reduction in downstream processing costs. These hollow-fiber systems have a small footprint and require minimal facility support (only an electrical outlet and CO 2 source, with no hard plumbing), so that multiple instruments can occupy a small space. Our 1,500- ft 2 GMP production corridor supports commercially available hollow-fiber bioreactors, equivalent to approximately 16,000 L of tank capacity (as shown in the photo on this page). The hollowfiber systems offer considerable flexibility: We can vary both the number of instruments inoculated and instrument run times to meet individual production schedules. Culture Medium: A third and often underestimated parameter for process development is the choice of a culture medium. We usually recommend some process development regarding medium selection. Typically, a discovery organization has maintained its cell line in basal medium supplemented with fetal bovine serum. One option for process development is to optimize the basal medium used for growth and production. We have found that slight alterations in a basal medium formulation can have substantial influence on the growth, productivity, and subsequent cost of large-scale production in a hollow-fiber system. Another option is to test cell performance in serum-free media to minimize regulatory concerns; in some cases, cells perform better in the serumfree media. Cells are thawed into a control medium (specified by the discovery organization), adapted to other media of interest, and inoculated in a controlled T-flask study. If cells do not grow or produce well in a T-flask using one particular medium, then that medium will not support good growth or production in a hollow-fiber system either. A microscale hollow-fiber bioreactor study is performed in parallel to address combinations of cell side and non cell side media for optimal growth at the lowest cost (3 6). The cell side medium used is always the complete medium (serum-free or basal medium plus FBS). The non cell side medium is either complete medium (expensive) or basal medium (inexpensive). If cells grow well with basal medium on the non cell side, then substantial cost savings can be realized at the clinical production scale. Table 1 provides data from an example project, in which our client used RPMI-1640 medium (Roswell Park Memorial Institute ) with 10% fetal bovine serum (FBS) to grow cells in T-flasks. The client had history of good growth and production with this cell line in a small-scale hollow-fiber system using complete medium on both sides of the fiber. However, the client had little time or funds to spend on process

4 Figure 3: Example microbioreactor screen followed by pilot-scale production. The client used an expensive serum-free medium to grow cells in T-flasks. A microbioreactor study was first performed to determine whether basal medium could be used on the non cell side; cells were inoculated at 5,000,000 per ml and harvested three days later. Results demonstrated that cells grew about as well with complete medium on both sides as they did with complete medium on the cell side and basal medium on the other (top). A 160-mL bioreactor was subsequently inoculated using serum-free medium on the cell side and basal medium on the non cell side, and the pump rates were increased according to glucose demand up to rates similar to that discussed in Figure 2. The total amount of antibody produced was 25 grams in 30 days (bottom). Extrapolation to the largest-scale XCell system (operating 20 bioreactors simultaneously) suggests a production capacity of 6 kg/yr for one instrument. (Abbreviations: GUR, glucose uptake rate; Ab, antibody) medium cost projections were estimated at $140,000 for phase 1 production. The cost of medium for the new serum-containing process was estimated at $19,000, and the cost of the more regulatory-friendly CD-ACF medium was estimated at $39,000. In either case, the client saw a $100,000 benefit in medium cost alone from a single $10,000, five-week study. Pilot-Scale Work: A pilot-scale production run in a Maximizer hollowfiber instrument is useful in four ways. First, the performance of conditions used to operate the system are verified. Second, material produced from this run can be used for purification process development (including virus removal validation). We typically generate 1 25 g in one month at this scale, depending largely on the cell-specific secretion rate. For antibodies, a typical purification protocol incorporates Protein A for affinity capture, followed by a low-ph hold (for viral inactivation), ion-exchange chromatography (for DNA removal), and nanofiltration (a virus removal step). Third, the purified material obtained can be used for research purposes or preclinical animal testing. Fourth, data from this run are directly scalable to the XCell, our largescale clinical production instrument. The pilot-scale studies do not add to our overall time to the clinic because the run is performed in parallel with master cell banking. However, if the client chooses not to perform such studies, then scale-up projections will be less certain, and clinical material may be needed for purification process development. That can cause a potential delay later in the project. Sometimes clients with set budgets will bypass the pilot-scale production step. However, those clients tend to be more flexible in the quantity of protein required for their clinical studies. An example pilot-scale run coupled with a microbioreactor study and a scale-up calculation is shown in Figure 3. CELL BANKING Before clinical material can be development. Therefore, we proposed a simple study evaluating one additional basal medium and one chemically defined animal component-free (CD- ACF) medium. The cell line adapted readily to the new media. In a T-flask, growth was as good as or better than in RPMI+10% FBS, but antibody production was higher in the two new media. In microbioreactors, cells required complete medium on both sides of the fiber when using RPMI and the CD-ACF medium. By contrast, cells did not require complete medium on both sides of the fiber when using the proprietary basal medium. When using the original growth conditions specified by the client, Circle Reader Service No. 131

5 produced, a master cell bank needs to be generated and tested for adventitious agents under GMP conditions. This is required to ensure safety and consistency in the subsequent GMP manufacturing process. A vial cell bank is typically produced, followed by a manufacturer s working cell bank (MWCB), which is generated from the MCB. The purpose of an MWCB is to reduce the number of vials required for manufacturing from MCB stock. However, an MWCB is usually not a priority for discovery organizations at this early stage. CLINICAL-SCALE GMP PRODUCTION AND PURIFICATION Ordering Supplies: The transition from process development to clinical production is often limited by the six eight week lead time associated with ordering large quantities of media. Once a medium has been chosen from the media screening study, the approximate volume required to complete clinical manufacturing is determined, and that amount is relayed to the media vendor for production scheduling. Time still remains for refining the order quantity as more information is gathered from pilot-scale production rates and purification recoveries. Production and Purification: Cells are thawed, scaled up in roller bottles, and inoculated into the XCell hollowfiber instrument for production of clinical grade material. The XCell is the only hollow-fiber instrument currently used to produce a licensed, injectable protein (ProstaCint, a prostate cancer diagnostic product from Cytogen, Typical production time is 60 days; however, the duration can be shorter or longer depending on specific production goals. When production is complete, the supernatant is clarified and purified on a campaign basis in one of the purification suites. Release: Upon completion of the manufacturing steps described above, batch records are consolidated, reviewed, and released. The batch records can be of considerable size, even for a small project. Original documents are retained by our company, and copies are provided to the client. PHASE 1 AND BEYOND Regardless of their size, most organizations find themselves performing substantial process development between phase 1 and phase 3. This includes wholesale changes in cell lines, media, and bioreactor operating conditions. As a project moves through phase 3 and beyond, more rigorous comparability studies are required to validate and implement any process changes. More substantial benefits will be realized down the line by making process development a priority early in the clinical evaluation timeline. REFERENCES 1 Hirschel, MD and Gruenberg, ML. An Automated Hollow-Fiber System for the Large-Scale Manufacture of Mammalian Cell Secreted Product. In Large-Scale Cell Culture Technology; Lydersen, BK, Ed. John Wiley & Sons: Hoboken, NJ, Gramer, MJ, et al. Effect of Harvesting Protocol on Performance of a Hollow-Fiber Bioreactor. Biotechnol. Bioeng. 1999, 65, Gramer, MJ and Britton, TL. Antibody Production by a Hybridoma at High Cell Density Is Limited by Two Independent Mechanisms. Biotechnol. Bioeng. 2002, 79, Gramer, MJ and Britton, TL. Selection and Isolation of Cells for Optimal Growth in Hollow-Fiber Bioreactors. Hybridoma 2000, 19, Gramer, MJ and Poeschl, DM. Comparison of Cell Growth in T-Flasks, in Micro Hollow-Fiber Bioreactors, and in an Industrial-Scale Hollow-Fiber Bioreactor System. Cytotechnology 2000, 34, Gramer, MJ and Poeschl, DM. Screening Tool for Hollow-Fiber Bioreactor Process Development. Biotechnol. Prog. 1998, 14, Michael J. Gramer, PhD, is the director of science and technology, Mark D. Hirschel, PhD, is the chief scientific officer, and Jae Sly is the sales and marketing manager for BioVest International, Inc., 8500 Evergreen Boulevard, Minneapolis, MN 55433, fax , info@biovest.com, BIA BIA Separations d.o.o. Separations RELATIVE ABSORBANCE (260 nm) Teslova 30, SI-1000 Ljubljana, Slovenia Phone: Fax: CIM CONVECTIVE INTERACTION MEDIA for Biochromatography An Evolutionary Approach from the Pioneers in Monolith Separation Technology! TIME (SECONDS) 16-mer 100 Anion Exchange Semi-Preparative Purification of a 16-mer Oligodeoxynucleotide on a CIM DEAE Disk Monolithic Column; Non-purified 16-mer oligodeoxynucleotide: red line, standards of 1 to 16-mer: blue line Optimized for the separation or purification of large biomolecules: such as pdna, proteins, peptides, and oligonucleotides Analytical and preparative chromatography in seconds Easy Scale-Up and Method Transfer without loss of resolution Conjoint Liquid Chromatography (CLC): Ability to combine different chemistries in one column allowing one step purifications, e. g. Affinity and Ion Exchange (Multidimensional Chromatography) Low Back Pressures even at High Flow Rates High Resolution: practically unaffected by flow rate High Dynamic Binding Capacity (Flow Independent): e. g. up to 60 mg of Protein or 10 mg of pdna/ml of support Chemically Stable: ph 1-14, can withstand 1 M NaOH Flow through channels have existed for billions of years in nature; they've just gotten faster with CIM supports! BIA Separations R & D, Production, QA, Contract Manufacturing % Buffer A Circle Reader Service No. 133