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.
Technology transfer of manufacturing processes for recombinant proteins frequently involves the reassessment of equipment
capabilities and corresponding process requirements. In this context, this article describes the transfer of a fermentation
process for the manufacture of Q-beta virus like particles (VLPs) in Escherichia coli (E. coli) from an external collaborator to Pfizer's cGMP pilot plant. In preparation for the technology transfer, establishment of
appropriately organized teams as well as effective communication at all levels were key (see Figure 1). These early teams
included both process development and analytical development scientists, since analytical assay support for an atypical product
such as the Q-beta VLP was considered important for the success of this endeavor.
Figure 1: Technology transfer team structure. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
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.
The fermentation process consisted of a seed culture stage and a fed-batch fermentation stage. The same yeast extract-enriched
medium containing glycerol as the primary carbon source was used for cell bank preparation, seed culture preparation, and
the fermentation batch phase (see Table I) (6, 7). For the fermentation fed-batch phase, two different fermentation feed media
were used; both were similar in composition to the batch medium, but contained either a high concentration of glycerol alone
or glycerol and lactose (see Table I) (6). Lactose was used as a carbon source and as the inducer.
Table I: Fermentation Medium.
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.