DO

REVIEW
Sustainable Reduction of Bioreactor Contamination
in an Industrial Fermentation Pilot Plant
Beth Junker,1* Michael Lester,1 James Leporati,1 John Schmitt,1 Michael Kovatch,1
Stan Borysewicz,1 Waldemar Maciejak,1 Anna Seeley,1 Michelle Hesse,1
Neal Connors,1 Thomas Brix,1 Eric Creveling,1 and Peter Salmon1
RY810-127, Merck Research Laboratories, Fermentation Development and Operations,
P.O. Box 2000, Rahway, NJ 07065, USA1

Received 25 August 2005/Accepted 1 April 2006

Facility experience primarily in drug-oriented fermentation equipment (producing small mole-

cules such as secondary metabolites, bioconversions, and enzymes) and, to a lesser extent, in bio-
logics-oriented fermentation equipment (producing large molecules such as recombinant proteins
and microbial vaccines) in an industrial fermentation pilot plant over the past 15 years is de-
scribed. Potential approaches for equipment design and maintenance, operational procedures,
validation/verification testing, medium selection, culture purity/sterility analysis, and contamina-
tion investigation are presented, and those approaches implemented are identified. Failure data
collected for pilot plant operation for nearly 15 years are presented and best practices for docu-
mentation and tracking are outlined. This analysis does not exhaustively discuss available design,
operational and procedural options; rather it selectively presents what has been determined to be
beneficial in an industrial pilot plant setting. Literature references have been incorporated to pro-
vide background and context where appropriate.

[Key words: culture purity, sterility, fermentor]

Bioreactors are one of the core components of bio-
pharmaceutical production (1). Volumes range from 300 l to
>10,000 l for animal cell culture and 40,000 l–250,000 l for
microbial culture (1, 2). The largest sterile fermentor ever
constructed had a working volume of 1,500,000 l (3). Loss
of sterility in bioreactors has been the most common cause
of process failure (1), and it has surpassed mechanical, elec-
trical or instrumentation problems that occur (4). In contrast
to chemical processing, there is no rework procedure to
recover. Thus, contamination compromises the entire batch
and equipment has to be shutdown for the subsequent fail-
ure investigation, upsetting either manufacturing or develop-
ment production schedules (5).
Consequences of contamination have included: (i) con-
version of nutrients to unwanted products, (ii) changed
broth conditions, such as pH, which degrade product and
adversely affect subsequent product formation, and (iii) en-
zyme formation which degrades product (e.g., penicillin in-
activation by penicillinase-producing microbes; 6–8). Some
facilities may choose to harvest contaminated batches after
investigating the contaminating organism (its type, level in
batch, and whether it produces an undesirable metabolite),
and its effect on online variables, titer, and downstream
* Corresponding author. email: beth_junker@merck.com
phone: +1-732-594-7010 fax: +1-732-594-7698
purification (9). Rarely are contaminated batches processed
if the contaminant is a fungus since fungi may produce low
level toxins. Often, material from contaminated batches is
not utilized in the clinic, even if the contaminant was un-
noticeable at a macroscopic level. When processing of con-
taminated batches is undertaken, harvest titers and isolation
yields may be negatively impacted.
In most ancient fermentations (e.g., bread, wine, cheese,
soy sauce), operators exposed fermentation substrates to
chance infection in a haphazard fashion (10). The acetone-
butanol fermentation developed by Weizmann during World
War I was the first truly aseptic fermentation (8). Since then,
bioreactor contamination has resulted in substantial losses
of time, materials, and revenue (11). Its cumulative impact
has been underestimated, particularly in biotechnology (12),
for fermentation products ranging from proteins to second-
ary metabolites to organic acids. Consequently, one formi-
dable problem for biochemical engineers has been contami-
nation prevention (10). Too high a contamination rate for a
facility or individual fermentor can indict even non-contam-
inated batches prepared in that same location by suggesting
contaminants were present but not detected in culture purity
samples (i.e., false negatives).
Contaminating organisms also are known as adventitious
agents or non-host contaminants (13). Lack of contamina-
tion is indicated by terms such as asepsis and sterility (de-

251
scribing the lack of any culture growth) which are slightly
misused to indicate monosepsis (8). Culture purity is a more
descriptive term indicating growth of a single desired cul-
ture.
Each fermentation process is unique and must be evalu-
ated separately with respect to sterilization requirements
and tendency towards contamination (14). This tendency is
influenced by the nature of the fermentation/contaminant,
equipment design/operation, and microbiological process
controls implemented (7). Simply put: “What works for one
process may not work for others” (14). Susceptibility to
contamination is varied (14), based on the protective nature
of the growth medium or environment (15). Thus, equip-
ment, operational, or procedural problems may be masked
in one process and later appear in another process (14). A
particular process with a high proclivity to contaminate may
have to be abandoned or altered for reasons such as inability
to successfully sterilize its medium (14).
Specific cultivation factors decreasing the likelihood of
contamination for a particular process are (14, 15): (i) low
or high temperatures outside the range of 20°C–40°C, (ii)
low or high pHs outside the range of 5–8, (iii) low dissolved
oxygen, (iv) high osmotic pressure, (v) high fermentor back-
pressure (slight decrease), (vi) switch to lean, defined me-
dium from rich, complex medium, (vii) presence of potent
antibiotics or high solvent concentrations, (viii) high or very
low trace metal concentrations, (ix) high or very low sugar
concentrations, (x) high shear (slight decrease), and (xi) low
initial pre-sterilization bioburden. Media and operating
conditions of secondary metabolite fermentations support a
wide range of contaminants (i.e., molds, yeasts, bacteria)
(14), and broth is rarely self-protected by low pH or wide-
spectrum antibiotic production (8). Enzyme fermentations
also can be susceptible to contamination since they often
contain rich media at neutral pH with no antibiotic activity
(16).
Contaminants vary by product type. Common brewery
contaminants were of bacterial and yeast origin. The range
of contaminating organisms was limited since broth pos-
sessed low pH, lacked nutrients such as sugars and amino
acids, and contained alcohol and hop resins (17). Winery
contaminants consisted of Lactobacillis which produced
lactic acid instead of alcohol, Acetobacter which converted
wine to vinegar, and molds which imparted off flavors (14).
For continuous fuel ethanol fermentations (Saccharomyces),
addition of sulfite/hydrogen peroxide or antibiotics con-
trolled contaminants such as Lactobacillus (18, 19). In bu-
tanol and ethanol fermentations, both solvent presence and
anaerobic fermentation conditions protected against con-
taminants (10); product concentrations of acetone and sor-
bose alone were sufficient to prevent contaminant growth
(10). Yeast, gluconic acid, and citric acid processes were
self-protecting due to their low pH (6, 10).
Fermentation processes have increased in productivity
through extension of the idiophase by adding fed-batch nu-
trients, resulting in longer fermentations (20). During the
idiophase phase of antibiotic fermentations, contamination
varies widely depending on the antibiotic being produced
(14). Many antibiotic fermentations are more susceptible in
their tropophase (14).
To mitigate risk, preventing contamination and testing for
contaminants are complementary practices (13). Contami-
nation risks exist in both aging facilities that are susceptible
to mechanical failures, and new facilities that have opera-
tional unknowns. Between batches, there is a balance be-
tween decreased turn around time (thus maximizing produc-
tion) and increased time for preventative maintenance (thus
reducing failures) (21). Design, testing, and training each
have an impact (22). Strategies for minimizing and investi-
gating microbial (4, 14, 23, 24) and phage (24) contamina-
tion are available.
DESIGN AND MAINTENANCE APPROACHES
General fermentor design considerations Well-de-
signed and maintained bioreactors and associated systems
are important to achieving desired sterility and culture pu-
rity goals (2). Interestingly, many design features that typi-
cally are incorporated for sterility also are useful for con-
tainment (i.e., minimization of microorganism release) (25).
For previously installed fermentors, the ability to cost-effec-
tively change equipment design is limited; thus procedures
are changed to meet altered requirements.
For drug-oriented fermentors, the steam seal heat barrier
against the entry of contaminants is well known (6, 15).
Some initially felt that diaphragm valves were more suitable
for sterility (10), although constant steam service deforms
Teflon-backed EPDM diaphragms (2). In this facility, three-
piece ball valves were found to be suitable and were imple-
mented when possible for easy ball replacement (75% less
time) without the cutting and welding necessary for existing
two-piece valves. Piping was rearranged (i.e., valves added,
steam/condensate tubing relocated) so that lockouts for haz-
ardous energy control during maintenance (a United States
Occupational Safety and Health Administration [OSHA] re-
quirement) were localized to shutdown equipment and did
not impact steam seals of adjacent equipment. Drug-oriented
vessels also utilized an inverted “U” (gooseneck) vent line
configuration to avoid grow-back from non-sterile areas and
prevent fall back of foam or entrained broth; the pipe sur-
face was kept sufficiently heated via a concentric steam
jacket (8). Viscous cultures that spew (i.e., entrained broth
in exhaust air) may coat this vent line heater, reducing its
heat transfer and grow-back prevention.
The selected level of facility automation results in trade-
offs. Restricting automation and minimizing sequencing re-
quired operators in both the control room and field to ob-
serve the sterilization while making manual adjustments.
There was greater manpower required and potentially higher
variability, but reliability was increased if malfunctions un-
identified by alarm conditions were determined immediate-
ly by personnel.
General utility design considerations Both product-
contact (i.e., sparger air, steam) and non-product-contact
(i.e., instrument air, chilled water) utility services affected
contamination. The level of installed utility back-ups (e.g.,
sparger and instrument air compressors, chiller) matched
the acceptable facility risk.
Some published design principles for sparger air were
adopted in this facility (6, 21). Air compressor intakes were
elevated above and placed upwind from fermentor exhausts
and cooling tower mist. There was an estimated one-log re-
duction in live organism concentration with every 3 m of in-
creased elevation (21). Intakes were installed 9 m above
grade and fermentor exhausts were located at grade.
For this facility, after-coolers were shifted from the
vendor’s standard design of directly downstream of the
compressor outlet to downstream of the retention chamber.
Consequently, air in the retention chamber remained ele-
vated in temperature (26) but was cooled before reaching
dryers and fermentors. In another facility’s installation, com-
pressor after-coolers were removed entirely, and sparger air
reached fermentors at 120°C. The facility’s monosodium
glutamate fermentation was unaffected since heat was rap-
idly dissipated owing to low broth viscosity; this approach
was not recommended for heat-sensitive or viscous cultures
(21).
A retention chamber (5400 l) was designed with baffles
to extend the travel path of hot air in plug flow and insulated
to minimize the inlet/exit temperature drop to < 2°C (21). It
was installed after the compressors, utilizing heat of com-
pression to sterilize air even at low residence times of 15 s
(21). The actual temperature at the compressor outlet de-
pended on the ambient inlet air temperature, compressor
load, and cooling tower water temperature to the compres-
sor intercooler. In this facility, at 11,000 l/min and 2.8
kg/cm 2, the compressor discharge and retention chamber
temperatures typically were about 93°C. The retention cham-
ber residence time ranged from 20 s for > 16,000 l/min to
1 min for 5000 l/min.
Condensate was removed using heat exchangers and dry-
ers. Air then was filtered to remove desiccant before reach-
ing fermentors. Liquid present in fermentor air filters was
caused by (i) poor system design or maintenance, or (ii) ex-
cessive pressure/temperature drop in the air supply header
(6). Dew point was monitored (i) when sparger air left the
utility building and (ii) just after it entered the fermentation
building. The dryer achieved the target dew point of below
−20°C, typically below −50°C at low demand. This dew
point changed up to ±15°C after flowing 150 ft to the fer-
mentation building depending on ambient temperature and
demand. A third, separate, air-reactivated, desiccant dryer
was used when maintenance was required on first two
switching heat-reactivated desiccant dryers, and a separate
cooling tower water-supplied heat exchanger was used
when maintenance was required on the refrigerated dryer.
These back-ups ensured uninterrupted drying of sparger air.
High quality dried and filtered instrument air ensured
automatic valve reliability. Replacing plastic instrument air-
lines with copper minimized leaks (particularly at fittings),
maintaining reliable and adequate instrument air pressure.
When the site instrument air supply was interrupted, a back-
up compressor and receiver tank prevented fermentor back-
pressure loss caused by automatic valves reverting to their
failure states.
General maintenance considerations A comprehen-
sive preventative maintenance (PM) program, including
product and non-product contact utilities, was implemented
to minimize downtime. This program included a post-exe-
cution review of all associated documentation to ensure fol-
low-through on identified problems. Specific elements with
high pay-back for the time and cost invested included: (i)
quarterly infrared trap surveys to identify traps that were
plugged, blowing through, or leaking, (ii) annual testing of
fermentor and transfer line valves for internal and external
leaks, (iii) annual replacement of vessel diaphragms, espe-
cially those in constant steam service, (iv) annual testing of
diaphragms on addition assemblies (valve arrays), both be-
fore and after repair, using pressurized air to detect leaks
from valves submerged in water, (v) annual internal inspec-
tions for larger tanks to ensure bolts were present and tight-
ened, and (vi) annual inspection of Ingold-style (Bedford,
MA, USA), Chemap-style (Sartorius, Allentown, PA, USA),
and threaded ports to remove burrs to prevent sticking.
A maintenance database tracked when work orders were
initiated, work was completed, and repairs were tested.
(Testing ensured repairs met expectations prior to returning
equipment to service.) The database permitted a quick re-
view of outstanding work orders for a fermentor prior to use
and tracking of repeat repairs to determine if additional
investigation or more extensive repair might be required.
Change control procedures for equipment and computer sys-
tems ensured changes were documented, communicated, and
appropriately evaluated for potential effects on contamina-
tion as well as validation.
Agitator The agitator seal was the main area of con-
tamination concern associated with the agitation system.
For steam-lubricated, top-driven seals, steam remained
applied to the seal, even when not in use. Thus, inadvertent
dry operation was avoided, although the constant heat expo-
sure enhanced degradation of the seal’s elastomer o-rings.
For condensate-lubricated, bottom-driven seals, residual
condensate remained between batches. After long periods
(2–3 mos) of inactivity, fermentors with bottom seals under-
went sterility testing.
The extra cost of high temperature fluorinated elastomers
(Kalrez, Dupont, Wilmington, DE, USA) on both double
mechanical seal faces was justified by seal replacement
costs owing to premature elastomer degradation. Seal fail-
ure, due to external or internal leakage, was determined
visually and by measuring air pressure decay rates. Since
most seals leaked slightly, acceptable and non-acceptable
leakage rates were quantified.
A two-to-three year typical seal service life was estab-
lished based on tracking seal failures. The relative standard
deviation (rsd) of the average seal life was high at 48–94%;
thus trends in seal longevity with scale were difficult to
identify. Investigation of fermentors with more frequent seal
failures often revealed problems such as worn bearings in
the mechanical drive. Owing to high cost, seals were not
proactively replaced every three years, despite probable re-
duced risk of additional damage to agitator stub shafts. Also
owing to high cost, gear boxes were not rebuilt regularly
unless excessive vibration was noted in predictive mainte-
nance tests; higher vibration caused shaft runout and even-
tually seal leakage. However, proactive foot (steady) bear-
ing replacement for 15,000 l fermentors was cost-effective
every 1–2 years.
Instrumentation Key instrumentation influencing con-
tamination-free operation included fermentor back-pressure
 J

TABLE 1. History and type of probe failures

(a) DO probes
Total failures Failure type (%) Repeats
Year Failure/total % Membrane Polarization Sensor Transmitter Cabling Other (%)
2004 5/199 2.5 50 0 40 0 10 0 0
2003 9/232 3.9 66.7 0 27.8 0 0 5.5 11.1
2002 21/271 8.5 50 5 15 5 17 6 23.8
If evidence of more than one reason for failure suspected, each reason is counted on a fractional basis.
(b) pH probes
Total failures Failure type (%) Repeats
Year Failure/total % Sensor Transmitter Cabling Dehydration Other (%)
2004 1/199 0.5 0 0 0 0 100 0
2003 5/232 2.2 80.0 0 0 0 20.0 20.0
2002 6/271 2.2 33.3 0 16.7 16.7 33.3 0

and temperature instruments which were critical to steriliza-
tion reliability. Pressure gauges were verified against pres-
sure transmitters at 1.1–1.3 kg/cm2, confirming their accu-
racy should transmitters become suspect. Resistance tem-
perature detectors (RTDs) were characterized at three tem-
peratures (0°C, 65°C, 130°C) using an oil bath (27). Culti-
vation (15°C to 45°C) and sterilization (0°C to 130°C) dual
range RTDs were tested for agreement at growth tempera-
tures (28). (Dual range RTDs, used at both Merck [28] and
Eli Lilly pilot plants [29], provided greater accuracy at
growth temperatures.) The sterilization range temperature
then was checked during empty tank (advance) sterilization
(28).
Foam sensors were upgraded to models that were sili-
cone-sealed on the product-contact side to prevent culture
growth into the device (28). Differential pressure (DP) sen-
sors in broth contact were examined carefully for leaks of
sensing oil. For flange-mounted DP sensors, uniform torqu-
ing to 68 N-m and use of lock washers minimized loosening
which trapped broth underneath gaskets. Bolt tightness after
heating and cooling cycles was confirmed to demonstrate
that the torquing regimen was adequate. A similar strategy
was employed for DP sensors with sanitary clamp-style
connections. Switching to a consistent style for all stainless
steel bolts/nuts greatly facilitated these efforts.
A pre-batch pH/DO probe response checkout was con-
ducted to ensure reliability. pH probe sensors were replaced
every 6 months or 6 batches (whichever came first), dis-
solved oxygen (DO) probe membranes were replaced every
other batch, and DO probe sensors were replaced annually.
Special field storage holders were installed to protect probe
tips, after they were prepared for service and prior to vessel
insertion. DO probes were stored with a foam pad and cap
over the membrane; pH probes were placed in holders filled
with 3M potassium chloride solution.
During batch sterilization, the DO probe reading was
tracked according to the following expected behavior: First
the signal decreased to 0% saturation as the temperature
rose due to steam displacing air and oxygen becoming less
soluble. The signal then remained at a minimum level with-
out bounce during the sterilization hold time, rose when the
airflow started, and remained steady once fermentor tem-
perature stabilized. After sterilization, pH probe perfor-
mance was examined using off-sets between (i) pre- and
post-sterilization probe readings, and (ii) post-sterilization
probe readings and laboratory-analyzed grab samples. When
a post-sterilization failure of a DO or pH probe was de-
tected, the vessel usually was discarded.
Over 3 years, the most common reasons for probe failures
were compromised DO membranes and broken pH glass
sensors (Table 1a, b). These failures were large sterility
risks owing to potential release of non-sterile electrolyte.
DO probe failures were about three-fold higher than pH
probe failures. A numbering system was implemented to
track probes with repeat problems which were examined
more closely. A reporting system for pH/DO problems was
developed and communicated monthly to raise awareness.
Gaskets and o-rings To avoid inconsistencies associ-
ated with visual inspection and reuse, manway gaskets and
external o-rings on probes and plugs were replaced with
each batch. A cost-effective, disposable, single-use gasket
was identified that could withstand the constant steam-
traced manway installed on drug-oriented fermentors. The
selected material, a high temperature peroxide-cured ethyl-
ene polypropylene diene monomer (EPDM) gasket, was re-
quired to withstand >275°F for > 400 h and sometimes up to
600–800 h without splitting or substantially sticking to the
vessel manway.
Filters Post-use integrity testing of sparger air filters
was performed only if a contamination occurred and not
before use. Uniform torquing of larger-scale sparger filter
housings assured a uniform seal (2); smaller scale housings
utilized sanitary clamps which sealed readily after proper
alignment. Sparger air filters were single use (except the
19,000 l scale) since filter costs were low relative to lost
material and time due to failed batches. Post-batch visual in-
spection of filters being discarded ensured procedures were
not imploding or otherwise damaging filters.
The sparger air filter element was protected by an air pre-
filter and a steam filter (8). The steam supply valve to the
sparger air filter was leak tested to prevent wetting and sub-
sequent contamination. The steam filter housing drain was
Templstik’d during sterilization to ensure condensate bled
appropriately. The most severe operating conditions occurred
when using a wet filters; thus using dry conditions provided
an extra safety margin (30).
 Installation of filter housings and line sloping such that
moisture drained away from filters during sterilization was
critical; otherwise condensate collected and blinded filters
(27, 30). When air was applied and steam shut off at the end
of sterilization, sparger air filter (non-jacketed housing) clog-
ging caused low air flowrates and loss of backpressure. In
contrast, vent filter (steam-jacketed housing) clogging caused
high backpressure. Raising vent filter jacket steam pressure
to 15 psig during sterilization reduced clogging; steam pres-
sure was reduced to 5–7 psig during operation to prevent
heat destruction.
Training of maintenance personnel Training and
documented communication of procedures was necessary
for all maintenance personnel. Development of critical and
non-critical instrumentation calibration procedures, includ-
ing operational tolerances, ensured awareness of accuracy
requirements. Establishment of piping installation prefer-
ences was particularly important since this facility was the
only large-scale fermentation facility on site. For drug-ori-
ented fermentors, these preferences included minimization
of dead legs, installation of steam entries on the top of pip-
ing, installation of condensate removal legs on the bottom
of piping, smooth aligned manual welds, line sloping for
drainage, and 316 L material of construction. Daily reviews
of maintenance jobs ensured prioritization and completion
of the most critical repairs. When necessary, work was exe-
cuted such that hazardous energy control locks were safely
removed each day so equipment could be returned to ser-
vice during off-hours; this advance planning minimized use
of sterility-compromised equipment, particularly transfer
lines. Ensuring that support personnel knew the importance
and status of projects undertaken in the facility, as well as
the material generation purpose, had by far the highest im-
pact.
PROCEDURAL APPROACHES
Pre-batch set up Fermentor integrity testing has mixed
support in the literature. Some practitioners believed it un-
likely for a contaminant to move past air flowing from a
leak in a positively-pressurized vessel. Others felt that grow
back might occur since the leak was at ambient temperature,
particularly if a channel (i.e., a fluid layer) was present (31).
Having observed the latter phenomenon in this facility,
comprehensive integrity test procedures were developed us-
ing hydrostatic and air pressurization testing. Pressure hold
tests (i.e., less than 0.07 kg/cm2 loss over 12 h) also were
used for biologics-oriented fermentors, which had dia-
phragm valves on all inlet and outlet piping. Pre-batch in-
tegrity testing, along with a 7–10 d sterility test, typically
was conducted prior to (i) 19,000 l scale fermentations since
they were higher value owing to their large size, (ii) smaller
scale fermentations for which medium ingredients or bio-
conversion substrates were expensive, and (iii) using nu-
trient tanks idled for >2 months.
Pre-batch set-up (as applicable to fermentor type) in-
volved re-taping threaded national pipe thread (NPT) fit-
tings, replacing o-rings on probes and Ingold blind plugs,
replacing sanitary gaskets, as well as cleaning all ports and
grooves. O-rings were sprayed with 70% isopropyl alcohol
(IPA) in water to facilitate insertion and minimize damage.
Personnel were trained to identify problems encountered in-
serting plugs or probes (as well as filters) rather than forc-
ing them. This training reduced misalignment frequencies
which not only potentially caused contamination but also
damaged carefully machined tolerances and/or stripped
threads. Fermentor set-up not more than 48 h before ad-
vance-sterilization balanced the need to prepare in sufficient
time to address problems with inadvertent disturbance of
what was already set up.
Within 48 h of media batching, steam sterilization of the
empty fermentor, or advance-sterilization, was conducted at
1.3 kg/cm2 and 124.5 ± 1.0°C for 1 h without installation of
the sparger air filter (unless at the 19,000 l scale) and with-
out probes (since pH sensors showed degradation when ex-
posed to air for >24 h). Afterwards the fermentor remained
under air pressure until just prior to batching. Personnel
visually examined inside the vessel during set-up and just
prior to batching. Very often an agitator seal or head plate
o-ring leak was detectable simply by checking for unusual
amounts of accumulated condensate.
Although sometimes viewed as overcautious (8), other
facilities have conducted advance-sterilization prior to leav-
ing fermentors idle as a final cleaning step or after contami-
nation with spore-forming bacteria (15). Longer advance-
sterilizations were undertaken at some facilities after re-
peated contamination, when residual contaminants were
suspected to remain in crevices. Still other facilities have (i)
conducted advance-sterilizations with water for 1 h prior to
batching (29), or (ii) sterilized immediately after cleaning,
pressurizing the vessel with air until preparations began for
the next batch. None of these steps were undertaken in this
facility.
Monitoring Monitoring of fermentor operation began
with monitoring of sterilization. Sterilization profiles were
examined carefully and unusual occurrences documented
directly after the sterilization and before proceeding with
subsequent processing (2). Examples included difficulty ob-
taining airflow through the sparger filter post-sterilization,
foaming during heat up and/or sterilization hold periods,
and Templstiking failures. Documentation not only aided in
contamination investigation, but also identified process-spe-
cific problems.
During fermentor operation as well as directly before
sterilization, steam traps were checked at least daily (2),
typically every 8 h in this facility. Inexpensive bleed valves
were installed for easy draining/clearing of plugged traps.
Fermentor sight glasses were not usually cleaned using in-
ternal steam since there was a sterility risk associated with
the initial delivery of accumulated steam condensate in lieu
of steam (8).
Proper utility system operation was monitored using a
data acquisition system or manually via a utilities checklist.
Periodic reviews were undertaken to evaluate proper opera-
tion (e.g., elapsed time desiccant dryers were in service be-
fore triggering regeneration). Measures to undertake in the
event of utility failures were documented. These procedures
reduced the impact of product and non-product contact util-
ity outages and promoted uniformity of response. After a
utility failure, affected fermentors were evaluated to deter-
 FIG. 1. Relative monthly average contamination rate (typically few or no batches run in July) normalized to January value.
mine whether to abort. Evaluations were conducted using
on-line trends (if available) and visual monitoring of field
gauges during utility loss and subsequent restoration.
Post-shutdown facility start up In addition to overall
facility start up procedures, start up batch sheets were exe-
cuted for each fermentor after the annual maintenance shut-
down to evaluate certain PM work such as instrument cali-
brations and control valve operation. If repair of an in-
strument or control valve was needed, post-repair testing
was documented. Trends for multiple fermentors of similar
scales helped quantify tolerable limits for accuracy and off-
sets, and a data history was developed. These batch sheets
assisted in reducing the traditionally higher fall contamina-
tion rates (Fig. 1) and maintenance issues. Specifically, indi-
vidual fermentor integrity tests conducted annually reduced
accumulation of unrepaired leaks. Start up batch sheets also
were implemented after extensive maintenance to ensure
proper operation and that all disturbed components were re-
instated.
After shutdown, sparger air was blown through distri-
bution lines at maximum flowrates into each unpressurized
fermentation vessel for 8 h to remove any accumulated
moisture from piping. Sterility batches, a few at each fer-
mentor scale, were conducted as checkouts and as refresher
training. Finally, annual cleaning of all vessels and transfer
lines with sulfamic acid was conducted which removed ac-
cumulated hard water deposits.
Vessel and media sterilization procedures To mini-
mize bioburden, full cooling (6–8°C) was applied to drug-
oriented fermentors after batching but before sterilization.
Batching and sterilization occurred in a timely manner; typ-
ically, after starting the media mixing, six fermentors were
batched within 6 h, then sterilized within another 4–6 h. To
minimize concentrated sugar (e.g., 50 wt%) crystallization,
full cooling was avoided since bioburden increases were
much slower than in growth medium. Full cooling also was
avoided for soluble starch-containing media to encourage
starch dissolution; for this growth media bioburden in-
creases were not expected to be slow, however.
Using backpressure control, sterilization target tempera-
tures were achieved within 1°C. It was important to main-
tain positive pressure during sterilization (16). If present,
manual drains of steam and/or sparger air filters remained
cracked just enough to promote condensate drainage while
ensuring forward steam flow through the sparger line was
maintained. After sterilization, vessel pressurization with
sterile (sparger) air prior to cooling avoided vacuum upon
cooling (32). Thus, in this facility sterilizations were ended
by applying sparger air and steam together until sparger air-
flow was established, thus avoiding substantial backpres-
sure drops. Raising backpressure to 1.5 kg/cm2 prior to in-
troducing sparger air and cooling avoided foam, especially
in larger vessels. Other facilities shut off steam and start
cooling while permitting back-pressure to decrease as low
as 0.2 kg/cm2 before applying sterile air (33); the risk of
pulling in non-sterile air appears higher with this air appli-
cation regimen.
Preparation of inocula and mid-cycle additions In-
oculation and mid-cycle additions are two process steps
which potentially introduce open transfers either within bio-
safety cabinets and/or in the environment surrounding equip-
ment. Minimization of risks associated with these steps pri-
marily for drug-oriented fermentations was undertaken in
this facility. Regular cleaning and bioburden testing of
biosafety cabinets (especially cleaning under stainless steel
working surfaces) and regular viable and non-viable partic-
ulate testing of cabinet air quality were conducted. It was
less desirable (and not required for drug processes) to test
during actual open process transfers since the meter’s suck-
ing action likely disrupted laminar airflow patterns. Expo-
 S
sure plates also were not used since these plates added clut-
ter, interfering if placed close to working areas where they
were most relevant in detection. However, a segregated cul-
ture purity/sterility testing area was established to minimize
cross-contamination with seed cultures. Although difficult
to implement for a multi-purpose pilot plant, some produc-
tion facilities utilized separate inoculum rooms for working
with bacterial, Actinomyces, molds, and sterility testing to
further minimize cross-contamination (4).
Laboratory seed was prepared in two non-baffled Erlen-
meyer flask stages (1×250 ml flask transferred to 3–6 ×2 l
flasks), then pooled into one 4 l aspirator bottle per fermen-
tor. Replacement of 2 l side-arm flasks with aspirator bottles
containing bottom nipples (15) (i) avoided tipping of inocu-
lum bottles, requiring only gentle swirling to suspend cell
mass, and (ii) accommodated larger inocula of 2 l without
risk of wetting cotton plugs. Use of cotton plugs prepared
without forming too tight of a wad and wrapped in a thin
layer of gauze was favored. Some authors advocated pre-
sterilization of cotton (4), but this step was not adopted. For
walk-in incubator rooms (which did not possess high effi-
ciency particulate air [HEPA] filters) used for laboratory
seed cultivation, housekeeping was improved, specifically
via regular floor cleaning and removing stored equipment.
Bioburden was minimized successfully; viable particles
typically were measured at 0.0 CFU/ft3 and rarely reached
0.05 CFU/ft3. Non-viable particles (0.5 µ or larger) typically
ranged from 500 to 2500/ft3.
Lots of frozen bagged seed from an inoculated seed fer-
mentor were prepared and tested for culture purity, elimi-
nating future open handling steps (34). Frozen bagged seed
has been successful for twelve cultures thusfar (six fungal,
three filamentous bacterial, three yeast, and one Escherichia
coli).
Open transfers, used for drug-oriented fermentors, were
effective in non-classified, open process areas primarily due
procedural precautions. Its low bioburden of 2–15 CFU/ft3
viable cells was the result of regular cleaning of drain
trenches as well as resloping of floors to minimize standing
water. Puncturing of Chemap septa for inoculation and at-
tachment of acid/base and antifoam containers was success-
ful using large bore Chemap-style needles (Sartorius) with
flaming of needles just prior to septum penetration to facili-
tate entry.
Assembly preparation was aided using models and digital
pictures, avoiding complicated written descriptions or dia-
grams. A few additional sterilized components were pre-
pared so potentially compromised items were not used. A
laminar, HEPA flow, cool down area provided clean, low
bioburden air for pressure equalization during cooling of
autoclaved materials. Unused sterilized components were
discarded after two weeks; there was neither re-sterilization
nor re-use of consumables.
For drug-oriented fermentors, acid and base solutions
were prepared non-sterilely relying on the heat of mixing
concentrated acid or base with deionized water to self-steril-
ize. Careful aliquotting procedures were developed to en-
sure proper preparation of at least 25 wt% of sulfuric acid
and 25 wt% sodium hydroxide from concentrated stocks;
lower concentrations were not reliably self-sterilized. Auto-
clave sterilization of several 20 l jugs of acid and base was
considered a safety risk. Acid/base jugs were replenished up
to three times, then contents were discarded and jugs sani-
tized with dilute bleach before being refilled. All jugs were
discarded annually.
Pre-sterilization of nutrient feed solutions that were sub-
sequently in-line filter-sterilized over several days avoided
grow-through of contaminants, particularly gram-negative
rods. Pre-sterilized, disposable bags were used as nutrient
feeding containers to avoid repeatedly assembling and auto-
claving plastic jugs with numerous fittings. Filters were pre-
ferred on both the bag outlet and inlet (if not too expensive),
just on the outlet if material transfer into the bag was pre-
sterilized, or just on the inlet (least desirable) if pumping
material through an outlet filter was unreliable owing to
clogging or accumulation of bubbles.
Tank-to-tank transfers Hard-piped manifolds trans-
ferred culture and media using pressurized sterile air (16)
via dip tubes. Transfer piping was steamed constantly (26)
or steamed for 30 min at 130°C (in excess of required Fo
values), although lower times and temperatures (similar to
autoclaves) of 20–30 min at 120°C have been noted (10).
Steam was shutdown by closing condensate valves first and
steam valves second, proceeding from the receiving to source
tank. The source tank steam was last, followed shortly by
opening the source tank valve and beginning transfer.
During tank-to-tank pressure transfers, a higher pressure
was maintained in the source tank so broth did not back-
flow. The head pressure between the upstairs and down-
stairs portions of the facility (8.2 m elevation difference)
was considered as well as the fermentor hydrostatic head
for larger vessels. When re-sterilizing the line post-transfer,
steam was used to evacuate leftover material either (i) into
the source tank if it was to be discarded, or (ii) through the
transfer line drain of a nearby inactive fermentor if the
source tank was to be retained. Unidirectional evacuation of
line contents minimized problems such as condensate trap
pluggage. Periodic water flushing of the transfer line after
nutrients or inoculum had passed reduced extensive build-
ups on ball valves.
Pressurized transfers were conducted with appropriate
transfer line cooling depending on the process. Typically
cooling was accomplished by emptying the initial hot mate-
rial from the source tank to the sewer; thus long transfers
were avoided to conserve material. Another technique, back-
cooling with media from the receiving tank (6), was done
when available inoculum volume was low, and it was being
transferred into larger tanks. After back-cooling, operations
paused with a cooled line for a few minutes while pressur-
izations were reversed to move from backward to forward
flow; this step required valve integrity to minimize contam-
ination risk.
Careful attention to seed fermentor details (such as inocu-
lation, sampling, and transfer line sterilization) was more
important than the proximity of seed to production fermen-
tors (4). During seed preparation the fewest possible trans-
fers from seed vial to fermentor were desired (4). Post-in-
oculation mid-cycle additions (such as acid/base) were
avoided in seed fermentations since the time for culture pu-
rity evaluation typically was short (1–2 d). Using one seed
 J
tank to inoculate more than three production fermentors
also was avoided. In selecting seed tanks, batches that had
questionable culture purity results were eliminated. Suspect
seed tanks with faster growth or a noted equipment or pro-
cedural mishap also were not used. Alternatives to using
questionable seed were considered, such as inoculating one
production tank with acceptable seed then using that tank to
inoculate other production tanks (26).
Training and staffing of operations personnel
Training was particularly important when labor was gov-
erned through unionized collective bargaining with unex-
pected turnover based on seniority and new personnel arriv-
ing with no prior fermentation experience (35), as in this
facility. The ability to educate all personnel regarding best
practices was as important as documenting these practices
in readily accessible forms. For example, an approved refer-
ence summary table (based on SOPs) comparing slight pro-
cedural differences in sterilization across fermentor scales
was created. Ongoing training on revised/new SOPs and
batch sheets helped ensure familiarity and clarification of
procedures. This training often included both classroom and
field review of drafts, including actual practice runs, to im-
prove reliability and accuracy prior to finalization. Training
of new staff included monitoring their ability to execute
procedures.
Explanation of the reasons behind operational procedures
(2) generated a logical framework to assist personnel to
recall the order of execution. Procedures that explained
valves/line function as well as the valve/line number (if de-
sired) permitted a ready understanding of what the step ac-
complished. A balance was struck between avoiding signifi-
cant procedural variation by individuals or shifts (4) and
overly restricting when impact was negligible.
Sufficient staff in both the microbiology support labora-
tory and the processing area was available so that work was
efficient but not rushed. Scheduling at a reasonable pace rel-
ative to available personnel also allowed time for unex-
pected occurrences since the pilot plant environment was
process development and not manufacturing-focused. A form
for new processes, establishing to facility users what to con-
sider in planning, was submitted well in advance of the ex-
pected run to permit discussion and mitigation of potential
issues/risks.
VALIDATION AND VERIFICATION
Fermentor sterilization-in-place tests Two key con-
cepts for sterilization were evaluated and tested (2).
Thorough venting of air was confirmed by examining the
temperature/pressure relationship (6). The use of super-
heated steam was avoided in favor of 100% saturated steam
(2).
Steam-in-place (SIP) studies (using thermocouples with
spore strips in the vessel headspace and spore solutions in
the liquid where feasible), prevalent in biologics-oriented fer-
mentors, were applied to drug-oriented fermentors (36) in
this facility. Geobacillus stearothermophilus spore D-value
testing was conducted for various media and nutrients used
in biologics-oriented and drug-oriented processing. Relative
D-values of spores in the solution to be sterilized compared
to those in water were compared (37). Higher temperatures
were required to obtain adequate sterilization backpressures
of 1.1 kg/cm2 for batch sterilization of concentrated nutri-
ents due to their lower vapor pressures (37). Higher temper-
atures also were used for low working volume sterilizations,
especially of concentrated nutrients, to obtain adequate tem-
peratures in the vessel headspace. For various scales and
working volumes, sufficient sterilization temperatures and
times were established for water (high D-value relative to
growth media) and for concentrated nutrients (50 wt% cere-
lose with a low D-value and 50 vol% glycerol with a high
D-value, each relative to water) (38).
Batch sterilization times for growth media were 40 min
at ≥ 122°C for 280 l fermentors and 45 min at ≥ 122°C for
800 l–19,000 l fermentors based on these studies. In another
facility, temperatures exceeding 121°C and times exceeding
30 min were viewed as potentially overcautious but no stud-
ies were reported (8). Higher sterilization hold temperatures
caused a greater increase in Fo and lesser increase in Ro than
longer sterilization hold times (37). Batch sterilizations,
conducted at larger scales, realized additional kill by longer
heat up and cool down times. In another facility, these two
philosophies were used at the 120,000 l scale to replace a
cycle of 119°C for 45 min by a cycle of 122°C for 12 min
based on computer kill calculations (39); actual SIP map-
ping likely would have generated a more aggressive cycle.
Fermentor sterility (media hold) tests SIP effective-
ness for each drug-oriented vessel in this facility was con-
firmed by conducting three successful, successive, sterility
tests or inoculated batches. Sterility media consisted of
6 g/l yeast extract (autolyzed code 106; BioSpringer USA,
Minneapolis, MN, USA), 6 g/l cerelose, and 1 ml/l polypro-
pylene 2000 (P2000; Dow, Freeport, TX, USA), adjusted to
a pre-sterilization pH of 7.0. A low-end sterilization temper-
ature range of 122–123°C for 40 or 45 min depending on
tank size was used, followed by a 7–10 d hold period. The
cultivation temperature was 35°C, rather than 37°C, which
was less optimal for bacteria so as not to exclude fungal
growth, but high enough for timely bacterial detection.
At least one sterility batch was conducted after each con-
taminated batch, and scheduling was arranged to accommo-
date this step > 90% of the time. The sterility test sometimes
was skipped if the contamination cause was unequivocally
uncovered or known to have been eliminated. Two sterility
batches were conducted if the fermentor had inconsistent
performance, or if the corrective action effectiveness was
not discernable. If sterility tests of a drug-oriented fermen-
tor failed twice for unknown reasons, then a third test was
run with the DP cell sensor removed (as an undetected hole
was suspected) and replaced with a blank cover.
The annual average percentage of sterility batches in-
creased (5.7± 7.3% from 1Q1990-3Q1997 vs 15.6 ± 6.1%
from 4Q1997-3Q2004) as the facility’s annual contamina-
tion rate decreased (Fig. 2) since sterility batches were im-
plemented to proactively test equipment and procedures,
particularly if unusual culture purity samples were obtained.
Despite this percentage increase, the absolute number of
sterility batches remained fairly constant (rsd± 25%). An
overall decrease in lost time and effort due to contamination
was realized since sterility batches were simple and low risk
 S
FIG. 2. Relative contamination rate performance normalized to
1990 value.
relative to complex and high-risk process batches.
Autoclaves The validation of autoclaves and load
patterns was conducted using thermocouples and spore
strips/solutions. Measurement and monitoring of tempera-
ture and pressure inside the autoclave assured air was vented
and saturated steam conditions existed (6). Larger liquid
quantities or solids which settle have been observed by
others to require longer times to attain their sterilization
temperature (6). For larger liquid quantities and multiple
containers which became larger heat sinks/sources, the
autoclave chamber itself required longer both to attain ster-
ilization temperature and to cool.
Specifically for seed flasks containing water, validated
cycle times at 121°C were 45 min for 500 ml in 2 l Erlen-
meyer flasks and 25 min for 50 ml in 250 ml Erlenmeyer
flasks. Maximum flask loads were established; adjacent
flasks could not touch as this adversely affected heat trans-
fer. Flask sterility tests assured cycle suitability for non-sol-
uble complex media, containing solids such as cotton seed
flour (Pharmamedia; Traders Protein, Memphis, TN, USA).
Since glass vessels were not desired in the process area, 8 l
polypropylene bottles (Nalgene; Nalge Nunc, Rochester,
NY, USA) were filled with 6 l of P2000 for antifoam addi-
tion. Due to the lower heat transfer of plastics such as poly-
propylene versus glass (15), these bottles required notably
longer autoclave times of 190 min for adequate sterilization.
Filter and tubing manifold assemblies also underwent suc-
cessful post-cycle filter integrity tests. Finally, 10 ml of
water were added to 4 l empty inoculum transfer bottles
(and larger amounts to larger bottles) to facilitate internal
steam generation.
Cleaning Cleaning effectiveness had a large impact
on sterilization effectiveness. The accumulation of solids
compromised sterilization (2). A regular program of exam-
ining and modifying existing cleaning procedures for new
products proved more efficient than adopting a worst case
approach for drug-oriented fermentors in this multi-product
facility. An automated clean-in-place (CIP) system promoted
cleaning consistency for contamination-free operation (40),
but this facility did not possess one.
Post-batch fermentor cleaning was facilitated by adding
water to broth prior to its pasteurization at 80°C which was
necessary before discard. Pre-treating fermentors using high-
pressure water streams (120 kg/cm2 at 5 l/min) or a steam/
water mixture removed caked-on deposits located on the
upper sidewall, upper dish, and especially in the agitator
mounting flange. Removal of fermentor backpressure first,
followed by airflow reduction, prevented broth back up into
the sparger air filter if the check valve was faulty or was not
located prior to the filter. For biologics-oriented vessels
equipped with mass flow controllers rather than meters and
separate control valves as in drug-oriented vessels, back-
pressure was removed slowly. This action avoided the con-
troller opening suddenly and widely to compensate for the
sudden pressure drop and thus blowing media forward into
the vent line and its filter.
Water usage was acceptable using a flooding method to
clean smaller tanks and even for larger 19,000 l tanks since
they were used less frequently. Flooding, or filling the tank
with cleaning agent, permitted direct contact with the tank
dome and avoided shadowing associated with CIP spray
balls. Spray balls were less effective after longer 3–4 week
secondary metabolite processes since spray velocity was
insufficient to remove heavily caked-on deposits. Flooding
ensured crevices associated with bolts were cleaned in fer-
mentors without all-welded internals. Finally, it permitted
the cleaning agent to be maintained at its optimal tempera-
ture of 80°C (±10°C), under agitation at 75% of maximum
speed, for the 1–3 d soaking duration. Shutting off all fer-
mentor steam seals but steam to the agitator seal during
soaking aided cleaning. Cleaning and rinse solutions also
were passed through the fermentor internals by air-pres-
surization or recirculation using a pump. If there was re-
peated contamination or uncontrolled foaming, more exten-
sive cleaning was undertaken, such as flushing the cleaning
solution through the vent line. High boiling of the vessel
with Na2CO3 or Na3PO4 (high pH, metal-chelating agents)
and a germicide were applied in other facilities after re-
peated contaminations (4), although the chemical interac-
tion with stainless steel should be evaluated for the concen-
trations used.
A diagram of the hardest-to-clean locations, along with
guidance on usage of high intensity flashlights, standardized
visual inspections after cleaning. Caution was exercised if
the vessel was wet since it sometimes appeared clean when
it was not. Cleaning studies for the seed and production
stages of 17 drug products (11 fungal, 3 filamentous bac-
terial/actinomyces, 2 yeast, and 1 E. coli), 3 biologics prod-
ucts (1 yeast, 1 E. coli, 1 animal cell) and 6 nutrients at sev-
eral fermentor scales established the robustness of the facil-
ity’s cleaning strategy. Swabs revealed that Ingold-style
ports had the highest total organic carbon (TOC) levels, pos-
sibly due to the 70% IPA applied to o-rings to facilitate in-
sertion.

MEDIUM SELECTION FOR
DRUG-ORIENTED FERMENATIONS
Medium components to avoid for drug-oriented fermen-
tations due to contamination concerns were those ingredi-
ents that foamed on sterilization (e.g., oat flour, tomato
paste) or clumped on addition to water (e.g., meals, pectins).
Storage conditions for raw materials were selected to
avoid formation of clumps (41). Media ingredients foamed
to different extents during sterilization; cotton seed flour
(Pharmamedia) fared better than soy flour which fared
better than oat flour and tomato paste. Many common anti-
foams were not effective in reducing foam at sterilization
temperatures. There was significant risk of foam contacting
non-sterile portions of the fermentor either during medium
sterilization or subsequent fermentation (33). Foam also
lifted particles from the medium and deposited them on ves-
sel sidewalls where sterilization was less effective. Heat
treatment of media at 80°C for 5–15 min during the steril-
ization heating phase aided dissolution of ingredients and
enhanced germination of spores.
An in-line mixer (homogenizer) disrupted clumps, ensur-
ing solids were wetted sufficiently. Media was recirculated
through the mixer for up to 1 h (3–10 turnovers), then passed
once-through the mixer prior to transfer to fermentors. For
materials that did not dissolve, others found that suspension
in cold water usually prevented lumping (4). Solids were
purchased in a finely ground form (4, 15) and if this form
was not available, larger particles were sieved from meals
(42). When handling solids-containing medium, the fermen-
tor agitator was stopped only momentarily for manual level
measurements. Steam was blown up through the fermentor
bottom valve for 30 s during heating to re-suspend any set-
tled solids.
Direct charges of powders to fermentors without using a
media mix tank were avoided, unless powders were slur-
ried and internals rinsed thoroughly (42). Batching soluble
starch or high concentrations of sugars (i.e., 300–500 g/l)
into warm water (50°C–60°C) promoted dissolution, as well
as avoided caking. Batch sterilization of oils for nutrient
addition was conducted by adding water (about 5–10 vol%)
to avoid dry heat sterilization conditions and lower spore
D-values. Some oils with higher levels of fatty acids also
aided in killing spores (20). Others have found that steriliza-
tion of complex medium containing calcium carbonate re-
quired considerably longer at pH ≥ 7 than at pH 5–6 (33),
perhaps due to lower spore D-values at lower pH values.
A memo documenting preferred media components from
a sterility, as well as cost, reliability, GMP, and corrosion
perspectives, guided media development from the earliest
stages. Use of modified production media, containing a re-
duced amount of any secondary carbon source, for the fer-
mentor seed stage avoided excessively rich, solids-contain-
ing, laboratory seed media that foamed upon sterilization
(43). For unusual situations, a sterility batch was conducted
to confirm procedures for both fermentors and seed flasks.
CULTURE PURITY AND STERILTY ANALYSIS
Culture purity, as well as sterility of uninoculated media
and nutrients, was ensured through all development stages
and for all production processes (13). To reduce production
losses, contamination was detected early (12). Sometimes
contaminants were latently present without causing macro-
scopic changes in on-line parameters (44), but were de-
tected in off-line culture purity tests. In other cases, due to
the time lag and discontinuous nature of culture purity test-
ing, on-line variables showed contamination before off-line
tests.
Although contamination reduction efforts centered on im-
proving equipment and sterile techniques, rapid contami-
nation detection methods for inoculum and seed tanks still
were required (35). Robust microbiological testing proce-
dures have prevented use of contaminated inoculum, pro-
vided information on contaminations promptly, and pin-
pointed the source (7). Permitting seed tanks to ferment at
least 20 h after inoculation gave adequate time for culture
purity assessment; partially cooling seed slowed growth when
necessary, very often without significant negative effects.
Contaminant detection has relied on off-line, time-con-
suming, classical microbiological methods (45). Over the
years, culture purity testing regimens in this facility were re-
fined and expanded to increase their sensitivity. Overly bur-
densome testing regimens were avoided. Incubation at both
24°C and 37°C detected both bacterial and fungal contami-
nants (33).
Sampling for contaminants Sterility and culture pu-
rity analysis was conducted daily in this facility, and in
other facilities once per shift (2), as well as after steriliza-
tion (AS), after addition of post-sterilization shots (ASshot),
after inoculation (AI), and just prior to harvest or discard
(final). For drug-oriented fermentors, these samples were
taken from the vessel’s sample nipple into pre-sterilized test
tubes (open transfer) with push on caps. A constant steam
bleed was used for steam-sterilization of this nipple (main-
taining the surrounding area hot between samples), and it
was cleaned regularly. One alternative was to submerge nip-
ples in disinfectant (10) and remove the steam bleed, but
this technique caused condensate to accumulate and limited
flexibility. For biologics-oriented fermentors, a contained
(closed transfer) system was used with a 0.2 µ filter-vented
glass sample bottle and an integrity-tested valve array to
provide a steamable connection.
Each laboratory seed train was sampled for culture purity
at the time of transfer to the subsequent stage: specifically,
the vial, first stage flask, and pooled inoculum from second
stage flasks. The vial underwent culture purity testing prior
to use, and culture purity was analyzed during laboratory
front runs used to establish seed train transfer timing. Pre-
transfer seed samples were taken about 8–16 h prior to pro-
duction fermentor inoculation, and immediately sub-cul-
tured. Pre-transfer samples were not taken prior to nutrient
transfers since nutrient tank contamination rates were his-
torically low at 0.7% (Table 2).
Testing for contaminants Direct microscopic exami-
nation to identify contamination required distinguishing
among culture, contaminant, and medium components (20).
Thus, a sub-culturing enrichment technique was imple-
mented at 37°C to supplement direct observation. Sub-cul-
turing usually did not reflect the sample’s original popula-
 TABLE 2. Summary of contamination rates as a function of process type
Number of batches Contamination rate (%)
Organism class
Process type (contaminated/total)
(number of cultures) Process type Overall
for process type
Secondary metabolite Filamentous bacterial/8 154/913 16.9 3.0
Fungal/19 273/2483 11.0 5.2
Bioconversion Bacterial/5 9/79 11.4 0.17
Yeast/4 8/365 2.2 0.15
Filamentous bacterial/4 5/49 10.2 0.096
Fungal/3 16/70 22.9 0.31
Constitutive/recombinant plasmid/protein E. coli/11 19/249 7.6 0.36
Yeast/5 2/214 0.93 0.038
Animal cell Various/6 5/31 16.1 0.096
Nutrients (including water and defoamer) N/A 6/749 0.8 0.12
Total Various 531/5202 N/A 10.2

tion distribution (12) since it selectively enriched for con-
taminants. Tryptic soy broth (TSB) detected most bacteria
(20). It was preferred by this facility over phenol red (PR)
dextrose broth which detected bacteria by turning yellow
with acidic contaminants as well as acidic production cul-
tures. Also in this facility, thioglycolate broth which de-
tected anaerobes was not used since anaerobes were not
likely to thrive in the oxygenated environment of aerobic
drug-oriented fermentations.
Media, nutrient, and broth samples were placed on test
within 36 h by inoculating 1 ml into 4 ml of TSB. TSB tubes
were prepared on-site in test tubes with push on caps; com-
mercial preparations had screw caps which were not ergo-
nomically attractive. To ensure adequate oxygen mass trans-
fer, inoculated tubes were shaken at 220 rpm for 15–30 h at
34°C–38°C. After shaking, tubes were used to streak tryptic
soy agar (TSA) plates, incubated statically for an additional
6 d, then read visually before discard for unusual turbidity
compared to similar tubes. TSA plates were incubated at
34°C–38°C for 3–5 d; a 5% CO2 incubator sometimes was
used for animal cell broth samples. At AS, ASshot, AI, final,
and twice per week during production, direct broth samples
were streaked onto Sabouraud dextrose agar (SDA) plates
and incubated at 25°C–29°C for 7–10 d. Occasionally, blood
agar plates were used in addition to TSA plates to obtain
faster contaminant growth, and potato dextrose agar plates
were used in addition to SDA plates if a fastidious fungal
contaminant was suspected. Tubes and plates were exam-
ined visually for turbidity and unusual colony formation re-
spectively on an interim and final basis. Viability has been
defined as the ability to form colonies; thus turbidity in a
TSB tube without subsequent colony formation was not
considered contamination.
Prior to clearing a seed fermentor for transfer, direct broth
samples as well as TSB subcultures were examined micro-
scopically. An automated gram stainer (Midas III; EMD
Chemicals, Gibbstown, NJ, USA/Merck KGaA, Darmstadt,
Germany) reduced multi-slide preparation variability. A
collection of digital photographs of typical gram stains, an-
notated with evaluations of the field, was developed for
reference and training. In some cases, it proved difficult to
determine whether the gram-stained rods observed origi-
nated from pre-sterilization medium bioburden inactivated
by sterilization or live bioburden due to a contaminant. Re-
incubation of the TSB sub-culture for a few more hours, fol-
lowed by re-examination and comparison to the prior field,
permitted qualitative assessment of whether live rods in-
creased in density. Fluorescence testing (LIVE/DEAD via-
bility kits; Molecular Probes, Eugene, OR, USA) using syto
9 green dye to stain all rods and propidium iodide to stain
dead rods (overwhelming the green stain) was investigated,
but was complicated by desired cultures often staining simi-
larly to contaminants.
A contaminated fermentor was declared when two con-
secutive samples, taken at least 18 h apart to permit correc-
tive action after investigation of the first contaminated sam-
ple, exhibited contamination. An unusual culture purity sam-
ple occurred when a sample was contaminated, but the sub-
sequent sample was not. Unusual culture purity samples (or
false positives) cost extra money in investigation and lost
product; an average of 0.25% false positives/year was esti-
mated for parental sterility tests with actual rates possibly
higher (46).
For the past six years, unusual culture purity rates were
tabulated by organism, process, and repeatability. Annual
rates ranged from 0.18% to 0.42% out of about 4000 to
5000 samples respectively, and no single process (fungal,
filamentous bacterial, nutrient, sterility) or contaminating
organism (gram-negative/positive rod, cocci, fungal) ap-
peared to dominate. Roughly a third of the unusual culture
purity samples repeated in retests with no clear trend with
process or contaminating organism. Unusual culture purity
events were most commonly found in direct broth samples
and TSB tubes rather than in plated medium, and they were
investigated in both the laboratory and pilot plant. Other
facilities have implemented isolator technology to reduce
risk of laboratory contamination during testing (47), although
published data concerning false positives is not available.
Contaminant identification Contaminant detection
has been based on contaminant cultivation on nutrient media
followed by identification via morphological and biochemi-
cal tests (47). Identification using fatty acid methyl ester
(FAME) analysis generated the most likely genus and spe-
cies matches for contaminants from this facility (Acculab
now Accugenix, Newark, DE, USA; Accugenix now uses
DNA sequencing.) The extent that the sample fatty acid pro-
file matched the mean profile of a database organism was
quantified by the similarity index: >0.5 indicated an excel-
lent match, <0.3 indicated a species not likely in the data-
base, and between > 0.3 and < 0.5 indicated a strain of the
species that differed significantly from the database organ-
ism. For contaminants with similarity indices <0.3, profile
comparisons indicated if contaminants were similar to each
other although dissimilar to database organisms.
Traditional culture purity/sterility detection methods
and their limitations Preparing microscope slides was
quick but not accurate, plating on solid media took longer
but was more accurate, and sub-culturing in enriched media
was even more time-consuming but most accurate (3). These
observations are consistent with experience in this facility.
There was a practical limit to contaminant detection using
these traditional methods. Limit of detection has been diffi-
cult to validate since bacteria show variability in growth and
are not usually homogeneously distributed in a sample; thus,
contaminants are unpredictable analytes compared to chem-
ical compounds (49). Others even have used stereomicro-
scopes to examine streaks on agar plates before colonies
were observed by the naked eye (4).
Sub-culture TSB detection limit testing was performed
(MDS Panlabs, Bothell, WA, USA), but costs were substan-
tial based on replicates required for suitable accuracy.
Quanti-cult cultures (American Type Culture Collection
[ATCC], Rockville, MD, USA) were selected, containing
10–100 colony forming units (CFUs)/0.3 ml inoculum,
which minimized manual enumerations. The detection limit
for three typical contaminants (Bacillus subtilis, E. coli,
Staphyloccus aureus) in sterile phosphate-buffered saline or
45 w/v% dextrose was >1.0 CFU/ml. Detection limits in the
presence and absence of desired organisms were predicted
to differ, however, necessitating process-specific studies. In
addition, further studies can verify holding times between
successive testing stages.
Alternative culture purity/sterility detection methods
The typical time required for sterility tests for injectables
is 14 d, and rapid methods are not currently listed in the
United States Pharmacopoeia (USP) (50). Conventional
membrane filtration has been used to obtain concentrated
cells from sterility samples followed by classic plate tests
requiring between 3–7 d (17). Although standard methods
for contaminant detection were reliable, they were per-
ceived as too time-consuming so the search for efficient and
rapid alternatives is underway (51). New techniques adopted
for contaminant detection need to be sensitive, selective,
and rapid (12). Methods were required to detect (i) a broad
range of microorganisms, and (ii) low contaminant levels
within high populations of desired organisms (13). Often,
even rapid microbial screening tests required contaminant
enrichment to meet the assay’s desired detection limits (52).
Commercially available, alternative contamination detec-
tion devices currently have focused on sterility testing. Gen-
erally, they have not been appropriate for culture purity test-
ing owing to difficulties distinguishing between desired and
contaminating cultures. Interestingly once techniques were
built into automated instruments, aspects of their metho-
dology lost transparency (51), making it harder to determine
applicability. A summary of available rapid methods for
culture purity testing, along with benefits and limitations, is
shown in Table 3. No one method emerges as widely ac-
cepted and implemented at this time.
CONTAMINATION INVESTIGATION
Investigation strategies The most successful efforts
to reduce contamination relied on thorough investigation in
an atmosphere of fact-gathering and avoidance of blame (2).
Communication with those closest to the process about po-
tential problems was conducted in a non-judgmental manner
to encourage openness (4). Anxiety associated with tech-
nique observations (particularly open handling steps), vol-
unteering feedback about what happened, or admitting a
significant error, was minimized. This atmosphere was
notably harder to create within a unionized environment,
where disciplinary action was associated with errors, or if
salaried personnel felt performance appraisals were im-
pacted.
When investigating a contamination, weak spots of the
process and equipment were broadly examined, avoiding
exclusive focus on what appeared the most likely cause or
favored theory. Attention focused not only on the produc-
tion fermentor, but also on the seed fermentor and auxiliary
piping/equipment (7). If the contamination was detected in
time, investigation began while the fermentor was still ac-
tive, visually examining set up and operation. A review of
relevant information concerning alarms and actions taken,
both planned and unplanned, was conducted.
More intensive contamination investigation then occurred
when the fermentor was discarded, cleaned, and disassem-
bled. Immediately after removal, pH/DO probes were visu-
ally inspected for damage and disassembled. Sparger air
and nutrient filters were inspected visually and integrity
tested. The steam supply valve to the sparger air filter was
checked for leakage. Fermentor integrity tests were con-
ducted to detect external and internal leaks, including valve
assemblies. Condensate traps were checked for pluggage.
Internal tank surfaces were visually examined for cleanli-
ness. Sight glasses, DP sensors, and foam probes, and any
associated gaskets, were removed, inspected, cleaned, and
reinstalled. Ball sections of three-piece ball valves and
vessel diaphragms were replaced. An agitator seal pressure
test was conducted for internal and external leaks. If there
was a repeat contamination, particularly of gram-negative
rods, the jacket was leak-tested using 90 psig air and a ves-
sel full of water. About the only time the contamination in-
vestigation was minimized or the sterility test omitted was
when a contaminated transfer was identified; even then,
some aspects of the investigation and possibly the sterility
test itself were performed to demonstrate contaminant re-
moval.
Assignment of most probable cause Common con-
tamination causes were seed, raw materials, inadequate ster-
ilization of equipment/air/media, inadequate procedures, in-
sufficient training, procedures not followed, lack of routine
PM, and bacteriophage (4). Other causes included construc-
tion materials, seals, valves, poor or overly complex design,
operator errors, instrumentation (calibration, automated valve
failure), sparger air, transfer/feed lines, contaminated in-
 TABLE 3. Summary of alternative rapid microbiological detection methods
Principle Method Technique Benefit/Limitation Detection limit Reference
Genetic markers/ Battery of negative Growth on ribose Recombinant strain 1 in 106 to 1 in 54, McFarland and
polymerase chain genetic markers- one minimal medium specific, sensitivity 1010 CFUs/ml of Swartz, Int. Pat.
reaction (PCR) set for each recombi- adjustable/Markers must recombinant strains of WO98/18955, 1998
nant host be engineered into strain same host cell type
Rapid detection of Specific DNA segment Rapid/Considerable sam- 105 to 106 cells in food 55
specific genes amplification ple preparation effort
Similarity of sample E. coli ribosome, Rapid (h or min)/Limited 2× 107 prokaryotic Webster, US Pat.
hybridization with that RNA-specific, DNA by primer specificity and cells in presence of 5348854, 1994
of known contaminants probe completeness of primer eukaryotic (animal)
library; gel identification cells
PCR via hybridization Amplification using of PCR product; inability 30 CFU/250 ml 17, 44
with DNA fragments universal primer pairs to differentiate between (0.12 CFU/ml) of lactic
of contaminants but directed to different live and dead organisms; acid bacterium in beer
not desired cultures conserved parts of designed for clinical and
prokaryotic rDNA environmental samples
gene
PCR-Advanced Real time PCR using 500 cells of Erwinia in 56, 57
nucleic acid analyzer TaqMan (homo- medium
(ANAA) geneous PCR using
fluorescence reso-
nance energy transfer)
and spectrofluoro-
metric thermal cycling
Triangulation identifi- Mass spectrometer- Single copy of 58
cation for the genetic derived base composi- contaminant genome
evaluation of risks tion signatures from specifically Bacillus
(TIGER) PCR amplification of anthracis in serum
highly conserved
regions of microbial
genome(s)
Light and radiometric Multi-parameter light Interchanges of differ- Real time, non-invasive, 1% B. thuringiensis 59
scattering (MLS) ent optical polariza- requires small liquid in E. coli (1–5%
tions resulting from volumes/Low sensitivity depending on system)
polarized light scatter- unless contaminants larger
ing than host organism-(less
Photon correlation Dynamic light scatter- likely in commercial fer- For 5% change in 45
spectroscopy ing to detect particle mentations) mean size: 1:71 ratio
size of E. coli to
P. aeruginosa; 10:1
ratio of E. coli to
S. cerevisiae
Scan RDI (Chemunex, Laser scanning system; 2 h detection/Selective 0.002 cells/ml in sterile 11
Paris, France) viable organisms lysis of mammalian cells medium (also used for
converted fluorescein- required so they did not animal cells)
based substrate into uptake fluorescent sub-
fluorescent product strate
Flow cytometry Blood cell lysis, stain- Rapid — <2 h, indepen- 10–100 CFU/ml, E. 60
ing of bacteria with dent of contaminant coli in buffer and blood
ethidium bromide growth/Electronic and
followed by stained hydro-dynamic “noise”,
bacteria detection ability to differentiate
using flow cytometry “target” from interference,
requires single cells to
pass through system
Flow cytometry Sample introduced to Rapid; counts very low 101–106 CFU/ml, 50, company litera-
(Advanced Analytical antibodies with fluo- numbers/Counts every- Pseudomonas and ture
Technologies, Ames, rescent tags (syto 62 thing — not just what Salmonella in media
IA, USA) stain), counts by focus- grows — giving higher and in presence of
ing on fluorescent numbers animal cells
events
BACTEC bacterial Sealed vial of 14C in- Faster than tubes and Linear trade off 35
growth promoter oculated with sample, plates, established medi- between bacteria
(Johnston Laboratories; 14
CO2 measured in cal technique/Non-growth detection time and
Cockeysville, MD, ionization chamber promoting medium or detection limit (100
USA) centrifugation minimized CFU in 9 h, 105 CFUs
background readings from in 6 h; sterile medium)
desired cultures with
mixed results, radioactive
substrate
 TABLE 3. (continued)
Principle Method Technique Benefit/Limitation Detection limit Reference
Immunological Binding with surface Fast immunogenic Faster than growth > 5000 bacteria/ml 52
polysaccharides separation for enrich- methods/Only specific (Salmonella) in 24 h/1
present in typical ment followed by rapid contaminating organisms pathogen in 25 g of
contaminants but not enzyme immunoassay can be targeted food
desired cultures
Chromatographic Gas chromatography in Uses muramic acid and Versatile, computerized 300 ng dry bacteria 12, 48, 61
conjunction with mass characteristic cellular evaluation/Profiles 500-fold bakers’ yeast;
spectroscopy fatty acids affected by growth phase 0.25% (w/w) Staphylo-
and substrate; challenging coccus and Bacillus,
to distinguish between 0.5% Streptococcus,
contaminant and desired and 1 ppm — 1%
culture unless isolated Enterobacter cloacae
— all in Leuconostoc
Different retention 260 µ id and 92 m Independent of contami- No actual cell systems 62
times of various size length, particle de- nant growth/Tested only tested
particles in empty tected by UV spectro- with particles; size differ-
column photometer ence required; unable to
distinguish live from dead
Morphological Differentiation of gross Varied agar media and Cost-effective/Sensitivity; < 100 CFU/ml in Paul Duncan,
colony morphology incubation tempera- time required for growth; > 109 CFU/ml recombi- personal communi-
and pigmentation tures; growth differ- development required for nant E. coli cation
ences substantially different < 100 CFU/ml in
desired cultures 5× 105 CFU/ml recom-
binant yeast
Phenotype-based Varied media formula- 4–6 bacterial or mold 13
enrichment/selection tions based on carbon CFUs/108 P. pastoris
source as selective cells
agent
On-line Adaptive computer On-line measurements Needs little prior process 1% (w/w) E. coli in 63
algorithm of fermentation knowledge; algorithm ad- yeast culture
parameters justable to be more or less
conservative/Challenging
to define contamination
alarm triggers; requires
suitable on-line measure-
ments
Artificial neural net- Contamination when Neural networks able to No direct testing done 64
work model real time process data model non-linear pro- to detect contamina-
varied ±25% from cesses/Training of mathe- tion.
ideal data continu- matical model required
ously for 30 min
eNose (model 4000; Electronic nose using “Real time” — does not No detection limit 65
Osmetech, Roswell, array of conductive require contaminant given for E. coli in
GA, USA) polymer sensors, growth on plates/Sensors M. carbonacea
responses analyzed become oxidized or
using pattern recogni- fouled; lot-to-lot sensor
tion variability
A recent summary of rapid microbiological methods also is available (66).

oculum, incorrect media sterilization, and inadequate proce-
dures (23). According to one author, the two most common
causes were equipment failures and operational errors (2).
After the contamination investigation was completed, a
most probable cause was assigned for tracking. In this facil-
ity, probable causes were grouped into four areas (Table 4):
sterilization failure, mechanical (equipment problem), oper-
ational (procedures not followed), process (procedures not
optimal), and contaminated inoculum or nutrient transfer
(detection not timely). Over the years, the operational cause
was eliminated since it often overlapped with sterilization
failure. A scoring system was used with a −2 for highly im-
probable, 0 if no information was available, and +2 for
highly probable. More than one category was utilized with
any number assigned (i.e., assigning a number to one cate-
gory did not exclude it from being assigned to another cate-
gory). One person was primarily assigning and one person
was primarily reviewing assigned numbers for consistency.
Although most every investigation revealed at least one
equipment problem, the contamination was only classified
as an equipment-probable cause if the problem found likely
caused contamination.
During the period 1Q1990 through 3Q1997, trends of
assignable causes indicated the highest percentage was un-
knowns (55.3%), followed by contaminated transfers (25.1%)
(Table 4). Substantial efforts then were implemented to im-
prove thoroughness of post-contamination mechanical check-
outs (to reduce unknowns) and pre-transfer clearing of seed
tanks (to improve detection methods). These actions not only
reduced the overall number of contaminated batches during
the period of 4Q1997 through 3Q2004, but shifted the high-
est percentage to equipment (54.6%) followed by unknown
 TABLE 4. Statistics for probable cause of contamination
Probable cause (number/% of period’s total)
Period Contaminated Failed
Equipment Process Unknown
transfer sterilization
Overall 118.5/23.9 69/13.9 30/6.1 20.5/4.1 258/52.0
1Q1990-3Q1997 110.5/25.1 39/8.8 29/6.6 18.5/4.2 244/55.3
4Q1997-3Q2004 8/14.6 30/54.6 1/1.8 2/3.6 14/25.5
If evidence of more than one reason for failure suspected, each reason is counted on a fractional basis.

TABLE 5. Statistics for type of contaminant

Type of organism (number/% of period’s total)
Period Gram-positive Gram-negative
Cocci Fungal Other
rod rod
Overall 58.3/30.7 82.3/38.1 31.3/16.5 9/4.7 10/5.3
4Q1994-3Q1997 45/33.1 55/40.4 21/15.4 8/5.8 7/5.1
4Q1997-3Q2004 13.3/24.2 27.3/49.7 10.3/18.8 1/1.8 3/5.4
Consistent data not available prior to 4Q1994. If multiple organisms identified for a single contamination, each organism was counted on a frac-
tional basis.

(25.5%) (Table 4). Further reduction in the percentage of
equipment-classified contaminations might be realized by
enhanced set-up or PM procedures.
Relation of contaminant type to probable cause
Both preliminary morphological/gram stain identification
(i.e., gram-positive or negative rod, cocci, or fungus) and
final identification (i.e., genus and species for non-fungal
contaminants; genus for fungal contaminants) of the contam-
inant were useful for investigation (42). Since final identifi-
cations typically required a few weeks, quick communica-
tion of preliminary identifications helped determine areas for
scrutiny.
General trends linking contaminant types to probable
causes were reasonably helpful although not absolutely firm.
Cocci originated from human handling such as direct con-
tact or breathing (14). Gram-negative bacteria sometimes
were indicative of a cooling water leak (14, 53), water
present in the inlet air stream (42), or incomplete filter ster-
ilization of non-sterilized mid-cycle additions (42). Gram-
positive bacteria often entered from non-sterile air (8),
owing to improper air filter installation, sterilization, or in-
tegrity (42). Gram-positive spore-formers possibly indicated
incomplete sterilization owing to large medium particles or
residual dried batch in vessel crevices (14). Yeasts some-
times originated from insufficient substrate sterilization (8).
One source of contaminating filamentous fungi and molds
was air (8, 14). In some cases, fungal contaminants also
originated from incomplete cleaning of crevices (42). Mul-
tiple contaminants were indicative of general sterilization
failure, often the air filter (14). There was no expectation
that the contaminant was a pure culture. Probable causes
were linked to identified contaminants for 51 contamina-
tions in this facility where a definitive probable cause was
ascertained; only some of these general trends were sup-
ported by the contaminant identification.
In other facilities, the most frequent contaminants were
spore-forming gram-positive or gram-negative rods; non-
spore-forming bacteria usually were absent except when en-
tering via jacket leaks (33). Fungal contaminations occurred
rarely (8). Fast-growing, spore-forming bacteria, such as B.
subtilis, were the most common contaminants in Actino-
mycetes and fungal broths (6). These basic trends were con-
sistent with the past experience in this facility (Table 5),
with levels of cocci and gram-negative rods at 40–50% of
gram-positive rods during the period of 4Q1997 through
3Q2004.
Contamination tracking Each contaminated batch was
counted in the overall contamination rate even if contamina-
tion arose from transfer of undetected contaminated fermen-
tor to multiple receiving fermentors. Each contamination
was weighted equally regardless of its volume, although
clearly the impact of a 19,000 l scale contamination was
higher. Both yearly and monthly contamination rates were
important to maintain a low overall failure level and avoid
monthly spikes >5%. There was little correlation between
annual contamination rates (Fig. 2) and numbers of batches
(Fig. 3). Typical batch complexity (i.e., preparations and
support required before, during, and after the batch) in-
creased during the period of 4Q1997-3Q2004; thus fewer
batches were run annually. Monthly and annual data sum-
maries were circulated to process supervisors, engineers,
upper management, and maintenance personnel, as well as
evaluated for trends (42). In addition, facility metrics were
highlighted to all, especially operators and mechanics, on a
regular basis.
The number of contaminations by scale and fermentor
were examined. Individual fermentor percentage contami-
nation rates were not readily obtainable from the database;
thus it was roughly assumed that within a scale each fer-
mentor was utilized more or less equivalently over the past
15 years. Within each scale, each fermentor’s individual rate
was within +2 SD of the average number of contaminations
for that scale. In fact, only three times was a fermentor’s
contamination rate outside the range of +1 SD; two of these
times were for a fermentor with a jacket leak subsequently
repaired. As the average contamination rate decreased, the
ability to highlight more problematic vessels increased.
The timing of contamination detection was tracked
 FIG. 3. Annual number of batches normalized to 1990 value.
(Table 6). About 65–70% of contaminations arose ≤ 48 h,
early in the cycle, regardless of time period. Examining
Table 6 in conjunction with Table 4 suggests that focusing
on equipment problems resulting in early contaminant intro-
duction might have the greatest impact in further contami-
nation rate reduction.
SUMMARY AND IMPACT
Contamination problems have delayed many fermenta-
tion plants from reaching their maximum production capac-
ities (10). Due to a contamination wave, some facilities
have discontinued the product associated with contamina-
tion and switched to another product (8). Some vessels only
were used for specific products because of unresolved con-
tamination issues that did not affect those particular prod-
ucts (53). Addressing pilot plant contamination was more
challenging owing to the research rather than production en-
vironment. Since a wide variety of processes were under-
taken, there was frequent switching of cultures as new strains
were obtained, and processes were less well-defined. A
lower contamination rate translated into less time and money
spent on investigations so these resources became available
for process development. Contamination in a fermentation
pilot plant impacted the corresponding isolation pilot plant
since it created subsequent gaps in that facility’s processing
schedule.
For this facility, the key change in performance over the
past 15 years was a progression in cultural outlook that
TABLE 6. Statistics for time of contamination detection
Time of contamination detection (number/% of period’s total)
Period
AS AI
Overall 39/7.9 79/15.9
1Q1990-3Q1997 32/7.2 74/16.7
4Q1997-3Q2004 7/12.7 5/9.1
Contamination time taken as time of first contaminated sample not of confirming samples. <48 h represents early phase, >48–120 h represents
mid phase, >120 h represents late phase.
occurred just prior to 4Q1997 — Denial: “It was a bad
sample.” Anger: “This happened because we don’t have ...”
Depression: “This process is always getting contaminated.”
Acceptance: “The monthly contamination report is issued.”
Action: “Our current contamination rate is not acceptable!”
A clear influence of culture purity on facility output was
evident, resulting in a direct increase in successful deliver-
ies and reliability of process development efforts. A proac-
tive versus reactive approach directly affected productivity
(avoiding transfer of contaminated batches), schedule (min-
imizing delays of subsequent batches while contaminations
were investigated), manpower (reducing time devoted to in-
vestigation), and maintenance (reducing amount of disas-
sembly and cleaning).
Published benchmarks for contamination vary consider-
ably. Despite problems in the pharmaceutical industry dur-
ing the 1950s, fermentors operated with < 5% contamination
(7). In the 1990s, PMs, SOPs, and teamwork were believed
able to reduce contamination frequency substantially less
than 5% (2). Rates of 5%–30% were thought to be realistic
with a contamination probability of 1 out of 100 acceptable
(8). Rates below < 1% were commendable and indicated
good performance (4, 8, 41), and rates of 2% were noted
for animal cell culture (8, 22). Specific contamination rates
for a facility rarely were published. One exception was the
contamination rate for monosodium glutamate production
which was reduced from 4.5% to 1.6% and then to 0.6% by
sparger air system upgrades, installation of a laminar flow
hood in the inoculum room, and repair of holes in the heat
≤ 48 h >48–120 h >120 h
204/41.1 95/19.2 80/16.1
176/39.8 85/19.2 75/16.9
28/50.9 10/18.2 5/9.1
exchangers of the continuous sterilization system (21).
Constant attention to detail is required for sustained con-
tamination reduction (53), and this commitment has been
undertaken in this facility. The ultimate tool in controlling
contamination is effective management and inter- and intra-
departmental team building (including training, communi-
cation, and trust) among team members with varied func-
tional backgrounds (2). Contamination awareness is main-
tained via written and verbal communication of the current
facility performance versus its metrics.
ACKNOWLEDGMENTS
This work represents the contributions of numerous individuals
associated with the Fermentation Pilot Plant (FPP) of Merck
Research Laboratories in Rahway, NJ, USA. These individuals
were the past and present process supervisors, research chemical
operators, mechanical coordinators, mechanical supervisors, trade
mechanics, engineers, and microbiologists. Special acknowledge-
ment goes to Dr. Peter Salmon who developed PARAFERM, a
customized Paradox database from which much of the information
for this paper was readily available.
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