Fermentation Process Technology Transfer for Production of a Recombinant Vaccine Component
A fermentation process for the manufacture of virus like particles (VLPs) in Escherichia coli (E. coli) was transferred from an external collaborator and rapidly implemented in Pfizer's cGMP pilot plant. Challenges faced in the transfer were meeting the high oxygen demand of the original process, and attempting high density cultivation of E. coli in a bioreactor system primarily designed as a seed tank for larger-scale mammalian and microbial culture. These concerns were overcome by an approach that combined process and equipment characterization, allowing suitable adaptation of the process to fit the pilot facility.
The Q-beta VLP is an antigen delivery platform designed to serve as a key component of therapeutic and prophylactic vaccines (1–4). Each Q-beta VLP consists of 180 copies of a single coat protein from the Allolevivirus Q-beta, which together form an icosahedrally symmetric particle stabilized by disulfide bonds (5). The first step of Q-beta VLP production involves cytoplasmic expression of the coat protein (Q-beta monomer) in E. coli. Subsequent processing and purification steps ensure the formation and recovery of mature VLPs. Technology transfer challenges related to the Q-beta VLP manufacturing fermentation process included: a change in scale, significant equipment-related differences, and the high oxygen demand of the original process.
Initially, the VLP fermentation process was transferred and implemented at the laboratory scale, which allowed further process characterization data to be collected. Following this, equipment characterization was undertaken to assess the capabilities of the cGMP pilot plant bioreactor system and compare it with the laboratory-scale system. Based on this information, required process adaptations were reviewed with our collaborators, and the process was successfully implemented in the cGMP pilot plant.
CELL LINE, CULTURE MEDIUM, AND ANALYTICAL METHODS
Strain and expression system
The strain used for production of the Q-beta VLP was an E. coli K-12 derivative that constitutively overexpresses the lacI repressor. The target protein, Q-beta monomer, was expressed under the control of the hybrid tac promoter induced by lactose or isopropyl beta-D-1-thiogalactopyranoside (IPTG) in a high copy-number plasmid. Plasmid selection pressure was maintained by a kanamycin resistance gene (6). The E. coli production strain was transferred to Pfizer as vials from a cGMP working cell bank (WCB) containing a low-density cell suspension in culture medium with cryoprotectant.
Samples were collected throughout the fermentation to assess culture growth and determine Q-beta monomer titer. Cell growth was examined by measuring absorbance at 600 nm (OD600), as well as determining dry cell weight with a 1 mL cell broth sample. Titer was determined by quantifying the amount of Q-beta monomer present in the soluble fraction of a lysed cell pellet using reverse phase-high performance liquid chromatography (RP-HPLC). Any fully or partially formed VLP present in the extract was first reduced to its monomer form by incubation with a reducing agent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was also used to ascertain monomer identity based on comparison with a standard. Cell-free spent fermentation media samples were used to measure glycerol and lactose concentrations. Glycerol content was estimated using a Nova Bioprofile 300 Analyzer (Nova Biomedical, Waltham, MA) and lactose was measured using a YSI 2700 Biochemistry Analyzer (YSI Inc., Dayton, OH). Off-gas analysis was carried out during the fed-batch fermentation runs using Tandem off-gas analyzers by Magellan Instruments (Limpenhoe, UK) to obtain an estimate of the oxygen uptake rate (OUR). After subsequent processing and purification, the quality of the intact Q-beta VLP was assessed by several methods, including analytical size exclusion chromatography (SEC). The titer and product quality assays were transferred from our collaborator or developed in house.
PROCESS TRANSFER TO LABORATORY-SCALE FERMENTATION SYSTEM
The objective of first transferring this process into the laboratory was to enable characterization of the fermentation protocol and allow the production of Q-beta VLP test batches for analytical characterization or development of the VLP purification process. It was understood that the VLP fermentation process implemented in the laboratory might require further adaptation to fit into Pfizer's cGMP pilot plant, as determined by the available equipment.
Original fermentation process
Process characterization and development
Laboratory scale fermentations were carried out in a 15-L working volume, computer-controlled Sartorius Biostat C DCU3 fermentation system. Operating conditions for the 15-L scale fermentations were derived from the original process conditions, taking equipment limitations and prior fermentation experience into account (6). For all laboratory fermentations, the dissolved oxygen (DO) set point was 20%, with cascade control by agitation (500–1200 rpm) and airflow (10–15 Lpm). The temperature and pH were controlled at 30 °C and 6.8 respectively. Operating pressure was increased with respect to the original process to increase oxygen solubility, because a pure oxygen stream to enrich the process air was unavailable.
A staged approach was followed for the technology transfer, which was intended to help understand the fermentation process requirements, such as oxygen transfer, and assess the laboratory fermentation system's ability to meet these requirements. This enabled adaptation of the original process for implementation in the laboratory without the use of oxygen, while maintaining productivity as close to the original process as possible. In practice, the staged approach was implemented by first performing uninduced batch fermentations, followed by uninduced fed-batch fermentations and finally induced fed-batch fermentations. Data from these runs drove discussions on process adaptation.
Batch fermentations were conducted to determine the biomass concentration after all the glycerol present in the batch medium was consumed. This data was required for calculation of the feeding profile. The batch fermentation medium contained 5 g/L glycerol as the main substrate for growth; however, additional substrate was also available due to the presence of yeast extract (6). At the 15-L laboratory scale, a starting batch volume of 10 L was used, and the batch was inoculated with 2.4% v/v shake flask preculture broth. During the batch fermentations, all glycerol in the medium was consumed in 7–8 h (data not shown).
Fed-batch fermentation with exponential feeding
Exponential fed-batch fermentations were designed to assess culture oxygen requirements and determine the maximum oxygen uptake rate that could be supported by the laboratory fermentation system. This would be observed on the DO profile as a sustained decrease in the DO value below the 20% set point. These fermentation runs included a batch phase as described earlier, followed by a fed-batch phase that involved the addition of glycerol feed medium at an exponentially increasing rate. The equation used to define the exponential feeding profile has been well documented in literature (6–9):
where, V(t) is volumetric flow rate of feed medium at time t (L/h), µ is specific growth rate (1/h), Vt,f is culture volume at feeding start (L), Xt,f is biomass (cell dry weight, g/L) at feed start (determined from batch fermentation), t is process time (h), tf is process time at feed start (7–8 h), Sf is concentration of glycerol in the feed medium (g/L), and YX/S is glycerol yield coefficient determined from batch fermentations (g dry cell weight / g of glycerol at the batch phase end).
Fed-batch fermentation with induction
PROCESS TRANSFER TO PILOT-SCALE FERMENTATION SYSTEM
The cGMP pilot plant fermentation system had a 100 L maximum working volume and had previously been used as a seed tank to support larger scale mammalian and microbial cultivation. Thus, it was expected that this system would have certain operating constraints that might require further adjustment of the laboratory-scale process prior to implementation. To prepare for the transfer, the pilot plant bioreactor was characterized and compared to the laboratory system.
The liquid phase volumetric oxygen mass transfer coefficient (kLa h-1), was experimentally measured to provide a quantitative estimate of the oxygen transfer capability of the system. Since agitation rate and airflow rate are the two most common means of controlling oxygen transfer and hence DO, it was decided to estimate kLa under different combinations of agitation and airflow rates, while keeping all other parameters the same. In order to maintain simplicity and transferability of the test protocol, the static gassing-out method was selected to estimate kLa, with water as the test medium (13, 19, 20). This test involved filling a vessel with water and alternately sparging with nitrogen or air to deoxygenate and re-oxygenate the test medium, respectively. The rate of oxygen re-absorption was recorded with an oxygen electrode and provided an estimate of the kLa through the equation:
where, C is concentration of oxygen in the medium at time 't' (mM/L), t is time in h, kLa is liquid phase mass transfer coefficient (h-1 ), and C* is saturation/equilibrium oxygen concentration in medium under experimental conditions (mM/L).
Heat transfer and heat load
To estimate and compare the cooling capacity of the fermentation systems at 30 °C, each vessel was filled with water, and the temperature set-point was repeatedly switched between 40 °C and 20 °C. All other parameters were set to operating values for the Q-beta VLP fermentation process. The recorded temperature profiles allowed calculation of cooling capacity, assuming the specific heat capacity of water at 30 °C as 1 kCal/kg –°C. Cooling capacity of the 15-L fermentation system was estimated at 21 kCal/min using 10 L water with 10 Lpm air flow, 755 rpm agitation at 30 °C (108 kCal/min–m2 of heat transfer area) (see Table II). The cooling capacity of the 100-L fermentation system was estimated at 60 kCal/min using 59 L water with 59 Lpm air flow, and 415 rpm agitation at 30 °C (90 kCal/min–m2 of heat transfer area).
The theoretical heat load was estimated based on OUR (9, 14, 21). For a rapidly growing culture using glycerol, assuming complete oxidation of the carbon source, and that all oxygen transferred into the liquid phase is used for the reaction:
where QH is the metabolic heat released in kCal/L–h, OUR is expressed as mM/L–h, and ΔHGlycerol is heat of formation of glycerol (0.397 kCal/mM). The heat load corresponding to an OUR of 250 mM/L–h was estimated at 5 kCal/min for an 11-L batch and 28 kCal/min for a 59-L batch.
Estimating water loss was considered important because the 100-L vessel did not include an exhaust condenser. Maximum theoretical water loss was estimated using a mass balance, assuming air entering the vessel was bone dry and air exiting the vessel was fully saturated with water vapor. Calculations assumed 59 Lpm air flow at 30 °C and atmospheric pressure. The water loss was also experimentally determined after holding water in the vessel under operating conditions for 16 h (59 L water at 30 °C, 420 rpm agitation, and operating pressure with 59 Lpm air flow). Theoretical evaporative losses were estimated at 0.12 kg/h for the 100-L vessel and actual water loss was measured at 0.06 kg/h after a 16 h hold step (see Table III).
Since the 15-L vessel used an exhaust condenser during normal operation, water losses would be negligible; however, for comparison, a theoretical evaporation rate of 0.019 kg/hr was calculated for the 15-L fermenter (10 Lpm air flow at 30 °C and atmospheric pressure). Actual water loss without the use of the exhaust condenser was measured at 0.0096 kg/h after a 44 h hold step (11 L water at 30 °C, 800 rpm agitation, and operating pressure with 10 Lpm air flow).
Transfer of the adapted process
An approach combining process and equipment characterization was used to transfer a high titer, fed-batch E. coli fermentation process for the production of Q-beta VLP rapidly and successfully from a collaborator to Pfizer's cGMP pilot plant. The early assembly of an appropriately staffed and sized technology transfer team that enabled efficient communication with the collaborator was key to the success of this endeavor. Based on a review of the fermentation process, high oxygen demand during the fed-batch phase was identified as an important issue, especially if the fermentation was to be reproduced without oxygen supplementation. The original fermentation process was rapidly transferred to a 15-L laboratory-scale fermentation system, while simultaneously collecting process characterization data. Subsequently, equipment characterization of the cGMP pilot plant and laboratory fermentation systems was undertaken. Based on these results, the original fermentation feeding strategy was modified to decrease the duration of the pre-induction feed phase and lower peak oxygen demand. The resulting fermentation process took advantage of the maximum oxygen transfer rate achievable in the pilot-scale fermenter, and successfully produced Q-beta VLP at a sufficiently high titer without the need for oxygen enrichment of the process air stream.
We would like to thank Cytos Biotechnology, Pfizer Vaccines Research Unit, Pfizer Bioprocess R&D Manufacturing and Analytical R&D Group, Michael Dupuis, Aparna Deora, John Amery, David Steinmeyer and Tom Warren.
Shamik Sharma* is a principal scientist, Allison Whalley is a scientist, Joseph McLaughlin is an associate research fellow, Frank Brello is a senior scientist, Bruce Bishop is a an associate research fellow, and Amit Banerjee is a research fellow, all in the department of Biotherapeutics Pharmaceutical Sciences, Worldwide R&D at Pfizer Inc, Chesterfield
MO and Andover MA. *To whom corresepondance should be addressed: Shamik.Sharma@pfizer.com
Article submitted: Nov. 18, 2010.
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