P.

Pastoris Fermentation Using A BioFlo® 110 Benchtop

Fermentor

Introduction
This Application Report is part of a series documenting culture growth in the
BioFlo 110. With appropriate vessels and control modules, the BioFlo 110 can
efficiently grow yeast and bacteria, as well as mammalian, plant cells and insect
cells.

Pichia pastoris:
Pichia pastoris is a methlotrophic yeast, which provides a unique expression
system for producing high levels of recombinant protein, including enzymes,
proteases, protease inhibitors, receptors, single-chain antibodies, and regulatory
proteins at various different levels. Pichia pastoris is also the only system that
offers the benefits of E. coli (cost effective, high-level expression and easy scale-
up) combined with advantages of expression in a eukaryotic system (protein
processing, folding, and posttranslational modifications).
A standard 7.5L BioFlo 110 Advanced Fermentation Kit was used to grow Pichia
pastoris in a fed batch fermentation. We used BioCommand Plus® supervisory
software to control the feed schedule, achieving 91.0 g/L dry cell weight (DCW).

Next, a BioFlo 110 Gas-Mix Controller was added, and the fermentation repeated
with oxygen supplementation of the sparge gas. This second run, described in the
APPENDIX (page 5), achieved a very high dry cell weight of 177.4 g/L. Neither
run was fully optimized, but the descriptions of procedures and materials, as well
as the data discussion will be useful to operators of similar fermentors.

The Fermentor
Vessel
The BioFlo 110 Advanced Fermentation Kit, NBS Catalog Number M1273-
1125, was equipped with a heat-blanketed 7.5 L fermentation vessel with nominal
5.7 L working volume. All BioFlo 110 fermentation vessels are configured with a
4-baffle stainless-steel insert, dual Rushton agitation impellers, and a high-speed,
direct-drive agitation system with mechanical face-seal. Dissolved oxygen and pH
probes (Mettler Toledo) are also included, as are a variety of items such as liquid
addition bottle kits (3), cables, tubing and clamps.

Control System
The four control modules included with the Advanced Fermentation Kit were used
for the first run.

Materials and Methods

Overview
This Pichia fermentation follows a well-established protocol in which glycerol is
the initial carbon source, and after a brief carbon starvation, we switch to a
methanol feed. The switch to methanol produces a metabolite of interest by
triggering the AOX1 promoter in genetically engineered Pichia. These are fed-
batch fermentations, since first glycerol and later methanol is added while the
culture is growing.

Control Program
We created a feed control program using BioCommand Plus software, NBS
Catalog Number M1291-0000. It turned on the glycerol feed-pump when the
dissolved oxygen (DO) level rose above 40%. Each time DO exceeded 40%, the
glycerol pump turned on; each time it fell below 40%, the pump turned off. 40% is
a high DO level, indicative of reduced metabolism due to carbon exhaustion. The
rationale for this strategy is that the DO increases due to reduced growth of the
cells, which is a result of nutrient depletion. Approximately one hour after a rise in
DO which indicated depletion of the supplementary glycerol, the program
automatically turned on the methanol feed-pump.

Inoculum
The inoculum was prepared using Pichia shake-flask growth medium:
Potassium phosphate monobasic (anhydrous) . . .11.5 g/L
Potassium phosphate dibasic (anhydrous) . . . . . . .2.7 g/L
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 g/L
10X YNB solution. . . . . . . . .. . . . . . . . .. . . . . . . 10% by volume
10x YNB solution consists of 67 g/L YNB without amino acids. The solution is
filter-sterilized and added to other media components after they are heat-sterilized
and cooled.

The inoculum was cultivated for 40 hours at 28°C in a rotary shaker (NBS model
G25) running at 240 rpm. Optical Density at 600 nm (OD600) was 10.94 at
inoculation.

Medium
The initial fermentor medium composition included:
Calcium sulfate dihydrate . . . . . . . . . . . . . . . . .. . . .0.93 g/L
Potassium sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . .18.2 g/L
Magnesium sulfate heptahydrate . . . . . . . . . .. . . . . .14.9 g/L
Potassium hydroxide . . . . . . . . . . . . . . . . . . . . . .. . .4.13 g/L
Phosphoric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .26.7 ml/L
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .40.0 g/L
Antifoam (Breox Foam Control Agent FMT 30) . . .1.0 ml/L

To allow space in the 5.7 L (working volume) vessel for components added after
sterilization, the initial medium volume was only 3.5 L. Post-sterilization medium
components included:
Trace Metals solution, PTM1 . . . . . . . . . . .4.6 mL/L
Base, to adjust the initial pH . . . . . . . . . . . .25 mL/L
Inoculum . . . . . . . . . . . . . . . . . . . . . . . . .. .200 mL
Glycerol* . . . . . . . . . . . . . . . . . . . . . . . . . .< 400 mL
Methanol* . . . . . . . . . . . . . . . . . . . . . . . . .<2 L
Base (to maintain pH at setpoint)* . . . . .. . .< 250 mL
(*) Added, as required

Pichia trace metals solution, PTM1 consisted of :

Cupric sulfate pentahydrate . . . . . . . . . . . . . . .6 g/L
Sodium iodide . . . . . . . . . . . . . . . . . . . . . . . . .0.08 g/L
Manganese sulfate monohydrate . . . . . . . . .. . .3 g/L
Cobalt chloride (anhydrous) . . . . . . . . . . . .. . .0.5 g/L
Zinc chloride (anhydrous) . . . . . . . . . . . . . .. . .20 g/L
Boric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .0.02 g/L
Sodium molybdate dihydrate . . . . . . . . . . . . . .0.2 g/L
Ferrous sulfate heptahydrate . . . . . . . . . . . . .. .65 g/L
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..0.2 g/L
6N sulfuric acid . . . . . . . . . . . . . . . . . . . . . . . .30 mL/L
Control Setpoints
Setpoints were keyed into the controller prior to inoculation and, except for DO
which remained high until culture was introduced, the vessel was allowed to
equilibrate prior to inoculation.
Temperature . . . . . . . . . . . . . . . . . . . . . . . 30°C
pH . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 5.0
Dissolved Oxygen . . . . . . . . . . . . . . . . ... . 30%
Agitation . . . . . . . . . . . . . . . . . . . . 300 - 1,200 rpm
(responding automatically to oxygen demand)

Dissolved Oxygen (DO) Control

The DO probe was calibrated at 0%, (obtained by briefly disconnecting the cable),
and at 100% (obtained using 1,000 rpm agitation and 5 L/m (1 vvm) airflow. After
calibration, DO remained at approximately 100% until inoculation.

An agitation cascade was selected in the controller to maintain DO at setpoint

through automatic adjustment of agitation speed. The agitation cascade increases
agitation speed with increasing oxygen demand. To set up the cascade, we used the
DO control display and keypad on the PCU to select:
Cascade: . . . . . . . . . . . . . . . . . . . .Agit
Minimum RPM: . . . . . . . . . . . . . . .300
Maximum RPM: . . . . . . . . . . . . .1,200

Nutrient Feed
Initial feed was 360 mL of 50% glycerol solution with 12 mL/L of PTM1 (trace
metals). BioCommand began this feed automatically when the dissolved oxygen
showed a sudden rise above the setpoint, a well-known carbonexhaustion indicator.
After all the glycerol was consumed, we allowed a brief starvation phase, then
changed the feed to 100% methanol solution with 12 mL/L of PTM1 (trace metals)
solution.

Glycerol . . . . . . . . . .Pump 2 of the 4-Pump Module

Methanol . . . . . . . . .Pump 3 of the 4-Pump Module
Transfer tubing . . . .Silicone tubing as supplied (1.4mm inner diameter and 4.8mm
outer diameter), P0740-2396
Vessel inlet . . . . . . . Triport adapter in the vessel headplate

Control Setup:
1) Pump 2 plugged into "Pump A" power-outlet of the Power Controller.
2) Pump 3 plugged into "Pump B" power-outlet of the Power Controller.
3) Pumps A & B: Manual mode, controlled by BioCommand

PH Control
We used liquid base to maintain pH at setpoint, relying on the acid-producing
culture to lower pH if needed. The pH control parameters were:
Base . . . . . . . . . . . . .ammonium hydroxide, 30% solution
Pump . . . . . . . . . . . .Pump 1 of the 4-Pump Module
Transfer tubing . .. . .Silicone tubing, as supplied, P0740-2396.
Vessel inlet . . . . . . . .Triport adapter in the vessel headplate.

Control Setup:
1) Pump 1 plugged into "BASE" power-outlet of the Power Controller
2) pH Control Selections: Multiplier = 25%
Dead-band = 0
PID values: factory defaults
Results and Discussion
The DO and agitation trend graphs (Figures 1 & 2) reveal the fermentation history.
We limited the carbon source in order to restrict growth to levels that non-
oxygenenriched air could support, which resulted in a healthy culture. Temperature
and pH were stable throughout the entire run.
The most important fermentor characteristics for highdensity cultures, such as
Pichia, are the fermentor's maximum oxygen transfer rate (OTR) and maximum
heat transfer rate. In dense robust cultures, the fermentor must:

1) incorporate oxygen at a high rate from the sparge gas into the dissolved oxygen
needed for metabolism. Additionally, OTR depends on agitation-motor power and
impeller design.

2) dissipate the heat of metabolism and agitation without allowing culture

temperature to rise above the growth optimum. Good temperature control depends
on cooling system design and coolant temperature.

Of course factors such as substrate concentration and metabolite build-up can also
be limiting, but these are often more controllable than inherent physical limitations
of the fermentor.

Conclusion
Pichia pastoris growth in the BioFlo 110 was successful. Culture density of 91.0
g/L DCW was achieved, and when oxygen supplementation was added (see
Appendix, page 5), cell density reached 177.4 g/L DCW.

Temperature control was excellent using unchilled (55°F) tap water as the coolant.
Nevertheless, fermentors with large-area stainless-steel heat exchangers, such as
the New Brunswick Scientific’s BioFlo® 3000, have an advantage in temperature
control at high cell density compared to systems with immersed coils (like the
BioFlo 110 used here); or when compared to systems that rely on waterjackets
made of glass. Glass has poorer thermal conductivity than stainless steel, but glass
jackets have larger surface areas than immersed coils. Both immersed coils and
glass jackets can work well, but the advantage of a fermentor like the BioFlo 3000
with a large stainless steel heat exchanger becomes significant at higher cell
densities or with higher coolant temperatures.

Overall, our protocol and the BioFlo 110 performed extremely well. The BioFlo
110 Advanced Fermentation Kit is a suitable instrument for culturing Pichia
pastoris. Furthermore, we expect that similar results can be achieved when using
BioFlo 110 fermentation vessels in other sizes (1.3, 3.0 or 14 L liters), as well as
when using a water-jacket configuration.

APPENDIX:
Effect of Gas Mix Controller and Oxygen Supplementation
A second continuous-batch was performed, this time adding the BioFlo 110 Gas
Mix Controller, , M1273- 3104, to demonstrate the impact of oxygen
supplementation on final dry cell weight. To maintain consistency, the media,
nutrients, inoculum, base, and control set-up was the same as the first run, except
for the DO cascade as listed below.
Dissolved Oxygen (DO) Control
Cascade……..Agitation and Oxygen

The cascade first increased agitation and then added oxygen gas as needed to
maintain the DO at setpoint.

Figure 4 shows that the DO declined steadily towards the 30% setpoint during the
first 10 hours, while agitation changed as required, to maintain the DO setpoint.
After reaching 1,200 rpm at ~24 hours, further oxygen demand went
uncompensated, causing dissolved oxygen to drop below the setpoint.