Lightnin: Fermentation: Critical Process Phenomena and New Technology Developments That Affect Yield and Productivity

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FERMENTATION: CRITICAL PROCESS PHENOMENA AND

NEW TECHNOLOGY DEVELOPMENTS THAT AFFECT


YIELD AND PRODUCTIVITY

IN
BY PRISCILLA KAUFMAN, PAUL KUBERA AND TOM POST

TO ESTABLISH A FOUNDATION FOR UNDERSTANDING RADICALLY NEW FERMENTATION PROCESS TECHNOLOGY,


THIS ARTICLE REVIEWS THE BASIC MECHANICAL AND PROCESS PARAMETERS THAT DETERMINE FERMENTATION

TN YIELD AND PRODUCTIVITY. AN OVERVIEW OF THE OPTIONS AVAILABLE TO AND EMPLOYED BY


PHARMACEUTICAL MANUFACTURERS IS PRESENTED IN RELATION TO THESE VARIOUS PARAMETERS. FINALLY, NEW
PROCESS TECHNOLOGY EMPLOYING UP-PUMP~NG HYDROFOIL IMPELLERS IS INTRODUCED. THIS TECHNOLOGY
TAKES AN ENTIRELY NEW APPROACH TO FERMENTATION THAT IS RESULTING IN SUBSTANTIAL IMPROVEMENTS IN
YIELD AND PRODUCTIVITY.

Introduction levels are required when oxygen transfer is the limiting step.
Aerobic fermentation is a big-chemical process used In this decade, tanks have increasingly been retrofitted to
throughout the pharmaceutical and biotechnology community extend power per unit volume to increase dissolved oxygen
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to create the active ingredients for numerous compounds. levels (especially in the critical phase) to improve yields.
Micro-organisms are fed nutrients and oxygen which enables To accommodate these volumes, mechanically agitated
them to reproduce and to metabolize those nutrients into other fermenters typically have a tank height to diameter ratio (Z/T)
by-products. Either the organisms themselves, their ranging from 2:1 up to 4:1, whereas air lift fermenters can
components or their by-products are then harvested through range up to 10:1. In full scale fermenters, that ratio is deter-
other down stream processes to create drugs such as mined more by US traffic regulations, which limit the maxi-
penicillin, Factor 8, hypolipidemic enzymes or other mum tank diameter that can be transported on US highways,
intermediate pharmaceutical products. than by particular mixing parameters. Uniformity as well as a
Because of re-validation and capital investment concerns, well mixed tank are much easier to achieve in more squat
many phannacoutical manufacturers use fermenters installed shaped-tanks. Tall tanks, such as those usually used in
in the 1950s. TraD/Tionally, these fermenters use sparge rings fermentation, tend to create individual mixing zones within
and multiple radial flow impellers known as Rushton turbines the tank that sigruficantly reduces blending.
to create conD/Tions that enable fermentation. However, Because of tank geometry, fermenters usually employ three or
recent advances in process technology have occurred. more impellers of similar diameter to achieve proper mixing.
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Regardless of whether a company needs to increase capacity or This number will vary; however, with the tank height to
simply replace or retrofit an older system, recent diameter ratio (HIT). Shorter tanks may have less impellers
improvements in mixing technology may enable while taller tanks require more. Moreover, the spacing of
manufacturers to substantially increase yields as well as impellers is critical to the process results. When impeller
improve productivity and product uniformity. spacing is too far apart, dead zones occur. These dead zones
contribute to the starvation of the micro-organisms which in
Mechanical Structure and the Impact on turn leads to a reduction in yield and productivity.
Fermentation Results To impart the power required for mixing, impellers with a
Full scale fermentation tanks typically range from 35,000 to smaller impeller to tank diameter ratio (D/T) need to spin
175,000 liters (9,000 - 46,000 gallons) and average about faster relative to impellers with larger D/T ratios. Increases in
75,000 liters (20,000 gallons). The agitators themselves range impeller speed; however, increase fluid shear within the tank
from 100 to 600 HP (75 - 450 kW), while most fall between and that shear can be detrimental to the micro-organisms in the
150 to 300HP(100 - 225 kW). High power per unit volume broth. Most often the D/T ratios range from 0.3 to 0.5. For
…… ……
2 PHARMACEUTICAL ENGINEERING SEPTEMBER/OCTOBER 1997

“THE PLACEMENT OF BAFFLES IS NOT TRIVIAL. POSITIONING AND DESIGN OF BAFFLES ALONG WITH
THE PLACEMENT, TYPE AND DESIGN OF HEATING COILS HAS A SIGNIFICANT IMPACT ON THE FLOW
PATTERNS AND, THUS, ON THE OVERALL PROCESS RESULTS."

bacterial and yeast fermentations which are not particularly differences within the tank greater than +3°C. Improving
shear sensitive, optimum D/T levels may be smaller, while mixing has been demonstrated to decrease temperature gradient
mycelial and, in particular, mammalian cell cultures, require to less than 0.2°C in industrial fermenters.
large D/T because of their shear sensitivity. Baffles are very important in the majority of fermentation
From an economic perspective, smaller D/T impellers have
fewer wetted surfaces and require less torque. This results in a
less expensive mixing system. On the other hand, large D/T
impellers require more torque and possess larger, heavier
wetted parts. Therefore, larger D/T impellers will cost more.
The cost of the impeller mixing system must therefore be
weighed against the value of the yield. Often the value of the
resulting yield improvements significantly outweighs the in-
creased cost of larger D/T impellers.
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processes. Baffles reduce the inefficient tangential velocity
component of all impellers to produce a more efficient radial
or axial flow. The placement of baffles is not trivial.
Positioning end design ofbaines along with the placement,
type end design of heating coils has a significant impact on
the flow patterns and, thus, on the overall process results.

Process Requirements
There are several concurrent mixing objectives in fermenta-
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Traditionally, gas (in the form of air or oxygen enriched tion including
air) is introduced into the system through a sparge ring or
open pipe that is placed beloved the bottom impeller. In most Solids Suspension: to suspend the media and biomass.
full scale fermenters, tanks are so tall that the hydrostatic head
of the liquid above the sparge rings requires the use of a Blending to uniformly blend the broth containing the media,
compressor to force air to the bottom of the tank. This has a micro-orgarusms, nutrients and any reagents as well as to
large impact on operating costs. maintain temperature and pH constancy throughout the broth.
Recent research has shown that splitting the gas flow to Blending is very dependent upon the mechanical arrangement
different locations within the tank can be beneficial to the of the fermenter as outlined above.
process. Placing the sparge ring higher up in the tank also
reduces hydrostatic head. This in turn allows for more cost Dispersion: to disperse the gas uniformly throughout the
efficient air blowers rather than compressors. For large fer- broth (and thereby increase gas hold-up), reduce the size ofthe
mentation plants, this results in a significant reduction in gas bubbles and prevent flooding.
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annual operating costs.


Heat transfer surfaces are placed between the impeller blade Mass Transfer: to dissolve the oxygen into the broth (gas-
tips and the tank wall to regulate the temperature of the broth. liquid mass transfer) enabling the dissolved oxygen to pass
These can be helical coils, vertical plates, or vertical coil through the ceil walls of the micro-organisms as well as
bundles - all of which have an impact on the flow pattern enable carbon dioxide flux in the opposite direction as the
developed in the fermenter. microorganisms metabolize (liquid-solid mass transfer).
It is important that agitators supply high flow over these heat
transfer surfaces to minimize hot and cold spots which will Heat Transfer: to facilitate the removal of heat created as a
have an adverse effect on temperature sensitive fermentations. result of cell metabolism as well as the heat generated through
In traditional systems, it is not unusual to see temperature the dissipation of mixing energy via viscous shear.
.…….
These basic mixing principles have been applied to fermenta-
tions in the pharmaceutical market since the 1960s. Research
on fermentation mixing technology tends to focus on mass
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transfer limitations because it has been assumed that mass


transfer is usually the rate limiting factor in fermentation.
Fundamental research on fermentation has always been done
on the small scale where mass transfer has indeed been the
rate limiting factor in the process.
Because blending is considered to be less complex than
mass transfer, it is the most overlooked parameter of full-scale
fermentations. None-the-less, blending efficiency has a sig-
nificant impact on yield and productivity. In fact, if the time
scale of mixing approaches the time scale of mass transfer, the
rate limiting factor shifts to blending. In full-scale fermenta-
tion, blending must be considered to maximize yield and
productivity.
All energy that is applied to a fluid mixer eventually appears
Figure 1. Baffled radial-flow turbine pattern. as heat through viscous shear. Ultimately, energy is dissipated

SEPTEMBER/OCTOBER 1997 PHARMACEUTICAL ENGINEERING 3

scale processes and affect particle sizes or cluster sizes of


200 microns or less. It is on the micro-scale level that both
mass transfer and diffusion occur.
The highest shear rates are in the impeller zone, generated by
the discharge velocity. This discharge velocity can be char-
acterized by an average impeller shear rate and a maximum
impeller shear rate. Regardless of impeller type, the average
impeller shear rate is only a function of the impeller speed.
Maximum shear rates, on the other hand, depend upon the
type of flow pattern generated. For radial flow impellers like
the Rushton Carbine, the maximum shear rate is a function of
impeller speed and diameter or tip speed. For axial flow
ungellers; however, the maximum impeller shear rate is also
only a function of speed.

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Upon scale-up, impeller speeds are always less than those
for pilot scale equipment. Therefore, average shear is always
less, whereas maximum shear rates always increase for radial
Figure 2. Time independent shear rate. impellers, but decrease for axial flow impellers. Thus, for full-
scale shear sensitive fermentations, it is not unusual to see
dissipated at the micro-scale level where it is transformed to better yields when using axial flow impellers.
heat as a result of viscous shear. In the case of fermentations
of long duration, this heat is significant and must therefore be
Coalescence
removed through heat transfer surfaces. Otherwise, the heat
Coalescence is the characteristic of substances coming to-
build up can be detrimental to the micro-organisms.
gether. In fermentation, this describes the tendency for smaller

as well as productivity gains.

Shear Rates
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Recent improvements in mixing technology discussed later
in this article affect blending, dispersion, heat transfer and
mass transfer as well as the interaction between each of these
processes and are resulting in significant yield improvements

Most common batch fermentations are bacterial, mycelial or


yeast based broths, and while all are affected by shear, myce-
bubbles to converge with other bubbles to form larger bubbles.
Most all pharmaceutical fermentation processes represent
coalesce systems.
In studying aerobic fermentation processes, the gas-liquid
behavior of these processes is traditionally evaluated using
air-water models. While theoretically these models do represent
coalescing systems, in reality they do not effectively behave so.
Once the gas is dispersed in water, it does not rapidly coalesce
to create the "bubbling" and "surging" seen in real
lial fermentations are by far the most shear sensitive. More- fermentations. Moreover, salts and electrolytes are often added
over, scale-up development of mammalian cell culture and to the fermentation broths as part of the testing process, further
rDNA is limited by shear sensitivity. To understand how causing the medium to behave more like a non-coalescing
GH
shear can affect fermentations, average and maximum shear system. This has implications for the traditional way
must be understood. This is especially important for shear fermentation processes have been analyzed and for the actual
sensitive fermentations such as newer cell cultures where effect of shear on full scale fermentation broths.
processes are moving from pilot to production scale For dispersion processes where bubbles do not tend to
operations. This is because in conventional fermentation coalesce, the effect of the average shear rates and maximum
systems, while average shear rates decrease on scale-up, shear rates on bubble size would determine the ultimate
maximum shear rates increase, and that can have a dispersion. Therefore, if the process lasted long enough, arid
significant impact on the process results. most fermentation processes do last several days, the ultimate
Time independent fluid shear rate is defined by a velocity bubble size would he determined by the maximum shear rate
gradient which is the change in average velocity over a given around the impeller since all bubbles would eventually move
distance. Time dependent fluid shear rate is defined by through this zone.
instantaneous velocity gradients over a given distance. This suggests that in a water-air model which acts effectively
Usually, these are determined in the impeller zone as well as as a non-coalescing system, the ultimate air bubble size would
throughout the entire batch. These data are usually obtained be determined by the maximum shear rate around the impeller.
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with hot film aenomometry, a Laser Doppler Velocimeter or This is an inaccurate representation of most fermentations
with a Particle Image Velocimetry. which have higher viscosities.
When looking at time indeperident shear, as described by Because most fermentation processes are coalescing sys-
average velocities, actual velocity fluctuations are obscured, tems, they cannot be adequately understood by air-water
but it is this variability in velocities that can have a significant models which more reflect non-coalescing systems. In reality,
impact on shear sensitive fermentations. Therefore, to account fermentations broths are of significantly higher viscosity than
for this, velocity fluctuations also must be accounted for by is represented by water. Moreover, as the micro-organisms
calculating the root mean square (RMS) value of the fluctua- within a fermentation broth multiply, viscosity increases.
tions (obtained by squaring the fluctuations, averaging them Viscosity acts as a drag inducing action such that the
and then taking the square root). This is a useful representa- reduction or lack of shear, enables bubbles to increase in size
tion of time dependent shear. (ie to coalesce). In aerobic fermentations; therefore, air
Shear rates operate throughout the tank at different mag- bubbles will tend to coalesce, and there is an equilibrium
nitudes and scales. The shear rate determined by the average between dispersion and coalescence. In this case, the entire
velocities affects particles in the order of several hundred spectrum of shear rates within the tank become relevant.
microns. The fluctuating velocity shear rates deal with micro Because of the pressure differentials around an operating
…. ….
4 PHARMACEUTICAL ENGINEERING SEPTEMBER/OCTOBER 1997

impeller, air bubbles tend to migrate toward areas of lower


pressure and coalesce. In aerobic fermentations, large bubbles “ALL AXIAL FLOW IMPELLER TECHNOLOGY
can get caught behind the impeller blades. This is especially
pronounced in Rushton Turbines, but is a problem with all IN FERMENTATION AS WELL AS IN MOST ALL
down-pumping impellers as well. Newer technology discussed PROCESSES IS PREFACED ON THE CONCEPT
below follows the natural flow patterns of coalescing OF DOWN-PUMPING.”
mediums. This eliminates the opportunity for bubbles to
aggregate into larger bubbles and stall the impeller.
Flooding occurs when the gas that is pumped into the
fermentation broth prevents the impeller from mixing the high efficiency axial flow impellers. This allows for improved
broth. There is a propensity for all radial flow and down- blending which, depending upon the process, in turn leads to
pumping axial impellers to flood in gassed conditions. To yield improvements. Because axial flow impellers have rela-
counter the possibility of flooding, the impeller diameters tively lower power numbers than radial flow impellers,
must be sufficiently large (which traps air bubbles) or the significant reductions in energy costs also are realized.
speed at which they rotate must be sufficiently great (which

IN
increases average shear). Fluctuations in velocity increase,
and micro-level shear rises which can have a detrimental
effect on shear sensitive cultures.

Impeller Technology
Traditionally, Rushton-type radial flow impellers have been
the impellers of choice in fermentations because they are
considered to be good for gas dispersion. However, these
turbines have relatively high power numbers (they require

TN
more power than other impellers of comparable diameter at a
given speed) and exhibit a significant decrease in power draw
upon aeration. Because the mass transfer coefficient is depen-
dent upon aeration rate and power per unit volume, this
implies a reduced mass transfer potential.1
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Figure 4. Fermenter mixer employing A310 axial flow upper


Figure 3. Rushton turbine. impellers.

Moreover, while the purpose of the bottom radial flow In the late 1980s, further improvements in the yields of
aerobic fermentation were realized with the introduction of
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impeller has been to disperse gas as it enters the tank and to


decrease bubble size to increase the surface area of the gas- high solidity ratio hydrofoil impellers. These impellers have a
liquid interface, the upper impellers do not further decrease the high ratio of total blade area to total projected sweep of the
size of these bubbles (in non-coalescing fermentations). Rather, impeller. With large, profiled and angled blades, these impel-
their function is to maintain the dispersion and circulation in lers produce strong axial flow patterns while providing supe-
the upper part of the vessel. Finally, compartmentalization of rior gas dispersion. When high efficiency axial flow impellers
the flow patterns is a problem with multiple radial flow cannot effectively handle gas, high solidity ratio hydrofoil
impellers like Rushton turbines. This results in increased ring impellers offer an effective alternative. Because oxygen mass
time as well as poor distribution of nutrients and dissolved transfer efficiency is improved, yield improvements are real-
oxygen. ized.
In the early 1980s, it was recognized that the purpose of the
upper impellers was primarily to provide uniform blending and New Process Technology
to allow contact between the air, broth and nutrients so that All axial flow impeller technology in fermentation (as well as
mass transfer can occur. Because the upper impellers do not in most all processes) is prefaced on the concept of down-
contribute to air dispersion, they can often be replaced with pumping. The impellers all circulate the fermentation broth in
…… …
SEPTEMBER/OCTOBER 1997 PHARMACEUTICAL ENGINEERING 5

“EVIDENCE SUGGESTS THAT THE MAXIMUM SHEAR


RATE DOES NOT CHANGE SIGNIFICANTLY ON
SCALE-UP, AND THIS HAS MAJOR IMPLICATIONS
FOR ALL SHEAR SENSITIVE FERMENTATIONS.”

Full scale up-pumping systems also demonstrate a four-fold


decrease in blend time. The dramatically improved blending of
nutrients and reagents can contribute to yield improvement as
well.
By encouraging air bubbles to circulate in the direction

IN
which they are naturally prone to move, up-pumping hydrofoil
impellers do not provide a surface for bubbles to coalesce.
Bubbles cannot collect under the impellers. Bursting bubbles
can cause cell damage in mycelial broth. By improving disper-
sion and reducing coalescence, "bursting" and "surging" is
decreased, suggesting a more hospitable environment for
mycelial culture.
Finally, as stated earlier, in the scale-up of traditional
fermentation systems, the maximum shear rate increases while

TN the average shear rate decreases. Maximum shear rates of


hydrofoil impellers do not increase on scale-up. With up-
pumping systems, evidence suggests that the maximum shear
rate does not change significantly on scale-up, and this has
major implications for all shear sensitive fermentations.

Conclusion
Each fermentation process presents unique constraints whether
they be from the process result (the product), the process itself,
Figure 5. A315 impeller. validation concerns or capital expenditure. An understanding
of basic fermentation mixing principles and aerobic fermenta-
a downwards motion with the intent to hold the gas down and tion behavior will help manufacturers determine whether up-
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longer in the tank. Therefore, in aerobic fermentation, the pumping hydrofoil impellers, high efficiency axial flow impel-
impeller pumps against the flow of the rising air. However, lers or traditional radial flow impellers meet their specific
recent research suggests that using hydrofoil impellers in an objectives.
up-pumping mode can increase mass transfer, improve blend- To do so, impeller characteristics such as average shear and
ing and heat transfer and decrease blend time. This results in fluctuating velocity as well as environmental parameters such
decreased reaction times, improved nutrient utilization, im- as viscosity and coalescence must be considered. This ensures
proved product uniformity and purity and decreased mechani- that the fermentation system is properly designed to maximize
cal vibration. In the retrofit of actual systems, yield increases process performance.
of 20 to 40 percent have been obtained. As pharmaceutical companies continue to come under price
Several factors likely contribute to such phenomenal im- pressure in the 1990s, improvements in manufacturing that
provements in yield. In the case of airlifts, up-pumping en- result in operating profit growth will be critical to a company's
hances the natural flow pattern of the gassed system. In success. The potential for significant yield improvement
mechanically agitated fermenters, up-pumping impellers are through the use of up-pumping hydrofoil impellers in aerobic
not working against the gas stream, and in fact, they cannot fermentation is one means for manufacturers to improve their
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flood. bottom lines.


Unlike Rushton turbines which have much higher power
numbers and which exhibit a large drop off in horsepower References
upon aeration, up-pumping axial flow impellers do not 1. Caroline M. McFarlane, Alvin W. Nienow, "Studies of High
demonstrate a significant difference between gassed and Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 1.
un-gassed horsepower. As stated earlier, the mass transfer Review," Biotechnology Progress 1995, 11, 601-607.
coefficient is dependent upon aeration rate and power per unit
volume. This therefore suggests improved mass transfer 2. Klaas van't Riet, Johannes Tramper, Basic Bioreactor Design,
potential at equal un-gassed agitator power. New York, New York, Marcel Dekker, Inc. 1991.
Recent studies using up-pumping technology also demon-
strate improved heat transfer and temperature uniformity. 3. James Oldshue, Fluid Mixing Technology, New York, New
Temperature gradients throughout the entire tank were found York, Chemical Engineering, McGraw-Hill Publications Co.
to be less than +0.2°C. This is a significant improvement over 1983.
the temperature distribution seen in traditional radial flow
impeller systems in which flow compartmentalization is often 4. N. Kioukia, A.W. Nienow, N. Al-Rubeai, A.N. Emery, "Influ-
a problem. ence of Agitation and Sparging on the Growth Rate and
……….
6 PHARMACEUTICAL ENGINEERING SEPTEMBER/OCTOBER 1997

Infection of Inset Cells in Bioreactors and a Comparison ing, research and manufacturing experts that is responsible for
with Hybridome Culture,” Biotechnology Progress 1996, addressing Lightnin's activities within the pharmaceutical and
12, 779-785. biotechnology industries. Kaufman is a member of ISPE.

5. Kenneth S. Lally, James Y. Oldshue, Ronald J. Weetman, Paul Kubera received both his BS and ME degrees in chemi-
"Mass Transfer and Fluid Mixing in Fermentation," Pre- cal engineering from Cornell University. Kubera began his
sented at American Institute of Chemical Engineers, No- career as a research engineer with US Steel Corporation where
vember 12, 1985 Annual Meeting, Chicago, Illinois. he specialized in environmental control, solids-liquid separa-
tion and by-products processing. Kubera joined Lightnin 17
6. James Oldshue, "Mixing in the Fermentation Process," years ago as a research engineer. Since then, he has held
Annual Reports of Fermentation Processes, Vol 6 1983, various positions in engineering and research. His areas of
75-99. particular interest are gas-liquid mass transfer, biological

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processes, waste and water applications and minerals process-
7. J. Y. Oldshue, C.K. Chan, H. Zemke, K. Bruegger, "Fluid ing. Kubera is a member of the American Institute of
Mixing Variables in the Optimization of Fermentation Chemical Engineers and the North American Mixing Forum.
Production," Process Biochemistry Vol 13, No 11, 1978.
Dr. Tom Post received his chemical engineering degrees from
8. J. Y. Oldshue, "Some Scale-up Considerations for Fermen- the Swiss Federal Institute of Technology in Zurich, Switzer-

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tation Mixers," Advances in Bio-Technology, Vol 1,517 land where he completed his doctorate in 1983. From 1983-
-522, Proceedings of the Sixth International Fermentation 1987, Post was an associate senior investigator for SmithKline
Symposium, London Canada, July 20-25, 1980. Beecham where he was responsible for the scale-up of many of
SKB's new organic synthesis projects as well as the improve-
About the Authors ment of existing product lines. In 1987, Post joined Lightnin's
Priscilla Kaufman holds a BA from Wellesley College and an R&D group where he has focused on mixer process perfor-
MBA in operations and finance from Columbia University. mance. From 1990-1993, Post was the director of Lightnin's
Before joining Lightnin, Kaufman worked in various interna- German R&D facility in Munich, and in 1995 he was named
tional commercial positions for Lederle Laboratories and later Vice President of Mixing Technology at Lightnin in
HT
consulted for Fisons Pharmaceuticals and Bausch and Lomb. Rochester, New York. His most recent projects deal with
Currently, Kaufman leads a biotechnology team of engineer- coalescing and non-coalescing gas liquid dispersions.
…… Lightnin, 135 Mount Read Blvd., Rochester, NY 14611.
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