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.
Starting from a single cGMP WCB vial, a high-density research and development cell bank was quickly created for laboratory
use (see Figure 2). Frozen cell suspension from the cGMP WCB vial was used to inoculate a small volume of medium in a 250
mL shake flask; this culture was then used to inoculate a 3-L shake flask. When the culture reached the desired OD600, the
cells were harvested by centrifugation and resuspended at a high density in fresh medium containing glycerol (20% v/v) as
a cryoprotectant. Aliquots were frozen at –80 °C for long term storage. High density cell banks with cell concentrations of
the order of 1 x 109 colony forming units were used to minimize seed vial requirements per run and ensure consistency with respect to seed culture
Figure 2: Preparation of laboratory (non-cGMP) and cGMP cell banks for Q-beta virus-like particle (VLP) manufacture. Lab
cell bank was tested for bacteriophage and purity only; this cell bank was used at risk until test results were available.
The cGMP cell bank preparation did not include the Shake Flask 1 stage.
Original fermentation process
The Q-beta VLP fermentation process to be transferred from our collaborator was a high-density fed-batch cultivation process
that was inoculated using a shake flask seed culture. The fermentation itself consisted of three phases: a batch phase, an
exponential fed-batch phase, and an induction fed-batch phase (see Figure 3). The batch phase lasted until glycerol in the
batch medium was consumed, and was immediately followed by the exponential fed-batch phase, that involved the introduction
of glycerol feed at a preset, exponentially increasing rate. Exponential feeding was continued for a set duration to achieve
a target biomass concentration, at which time the glycerol feed medium was replaced with induction medium containing glycerol
as well as lactose, and the induction fed-batch phase commenced. The induction medium was fed at a constant rate, equal to
the feed rate at the end of the exponential feeding phase. The induction phase lasted a predetermined duration, after which
the culture was harvested. This protocol was repeatedly shown to achieve a high titer of the Q-beta monomer along with a high
biomass concentration (6). Significant biomass accumulation occurred during the induction phase despite the initiation of
recombinant protein production. An oxygen-enriched air stream was used to satisfy the high oxygen demand of the culture during
Figure 3: Feed profiles for Q-beta virus-like particle (VLP) fermentation. The solid line shows original feeding protocol.
Feed I was the glycerol feed added at an exponential rate. Feed II was the glycerol/lactose feed added at a constant rate
for induction. Dotted line shows the manner in which exponential feeding was extended for process characterization.
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).
It was observed that after about 8.5 h of feeding, to maintain the dissolved oxygen set point at 20%, the agitation and airflow
reached maximum values of 1200 rpm and 15 Lpm respectively, and that beyond this time the set point could not be maintained
(see Figure 4A). Taking this into account, the duration of the exponential feed phase was adjusted to 8 h for future laboratory
runs, which was shorter than described in the original process but deemed satisfactory to allow sufficient biomass accumulation
for induction. The OUR at this time was measured at approximately 230 mM/L–h.
Figure 4: Q-beta virus-like particle (VLP) fermentation at the lab scale. (A) Characterization experiments to observe process
oxygen requirements - dissolved oxygen and oxygen uptake rate profiles for fermentations conducted with the extended exponential
feeding. (B) Dissolved oxygen and oxygen uptake rate profiles for modified fermentation process with shortened exponential
feed phase to decrease peak oxygen demand. Colors show dissolved oxygen profiles for different batches.
Fed-batch fermentation with induction
These fermentation runs were conducted to complete the laboratory-scale fermentation process transfer and assess final Q-beta
VLP production with a shorter (8 h) exponential feeding phase prior to induction. The batch and fed-batch phases were conducted
as described earlier. At the end of the exponential feed stage, glycerol feed medium was replaced with the induction medium
containing glycerol and lactose. The induction medium was added at a constant feed rate equal to the feed rate reached at
the end of the exponential feed phase. The induction phase duration was maintained at 5 h as described for the original process.
The culture density increased significantly during the induction phase, and the cell density at harvest was more than twice
that at induction. The OUR decreased after induction and remained at approximately 150 mM/L–h (see Figure 4B). Some lactose
accumulated during the early part of the induction phase as the culture adapted to this new carbon source (data not shown).
As expected, the harvest cell density and final titer were approximately 80% and 85% of the corresponding values for the original
process, respectively (see Figure 5A). The specific productivity, calculated as grams Q-beta monomer per grams dry cell weight,
corresponded very well with the original process, and product quality after purification was also satisfactory (see Figure
5B and 6A).
Figure 5: Culture growth and productivity data at the laboratory scale. (A) Growth for the adapted fermentation and original
process. Original process is in red. (B) Productivity of the adapted process was comparable to the original process.
Fermentation process transfer to the laboratory was completed in approximately three weeks, including preparation of the development
cell bank. A well-characterized small-scale fermentation process that fully utilized the capabilities of the 15-L laboratory
fermentation system was now available to produce test batches of Q-beta VLP for development.
Figure 6: (A) Adapted process showed comparable product quality at the lab scale. Analytical size exclusion chromatography
(SEC) was one of several methods used to test VLP quality. SDS-PAGE was used to observe the monomer (inset). (B) Adapted
process showed comparable product quality at the pilot scale. Analytical SEC and SDS-PAGE were among the assays used to used
to check VLP quality (inset).