Sustainable reduction of bioreactor contamination in an industrial fermentation pilot plant

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 4, 251–268. 2006 DOI: 10.1263/jbb.102.251

© 2006, The Society for Biotechnology, Japan

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 molecules such as secondary metabolites, bioconversions, and enzymes) and, to a lesser extent, in biologics-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 described. Potential approaches for equipment design and maintenance, operational procedures, validation/verification testing, medium selection, culture purity/sterility analysis, and contamination 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 documentation 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 provide background and context where appropriate. [Key words: culture purity, sterility, fermentor]

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 unnoticeable at a macroscopic level. When processing of contaminated 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 acetonebutanol 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 secondary metabolites to organic acids. Consequently, one formidable problem for biochemical engineers has been contamination prevention (10). Too high a contamination rate for a facility or individual fermentor can indict even non-contaminated 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 contamination is indicated by terms such as asepsis and sterility (de-

Bioreactors are one of the core components of biopharmaceutical 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, electrical 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 failure investigation, upsetting either manufacturing or development production schedules (5). Consequences of contamination have included: (i) conversion of nutrients to unwanted products, (ii) changed broth conditions, such as pH, which degrade product and adversely affect subsequent product formation, and (iii) enzyme formation which degrades product (e.g., penicillin inactivation 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: [email protected] phone: +1-732-594-7010 fax: +1-732-594-7698 251

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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 culture. Each fermentation process is unique and must be evaluated 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, equipment, 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 backpressure (slight decrease), (vi) switch to lean, defined medium 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 widespectrum 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 possessed 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 controlled contaminants such as Lactobacillus (18, 19). In butanol and ethanol fermentations, both solvent presence and anaerobic fermentation conditions protected against contaminants (10); product concentrations of acetone and sorbose 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 nutrients, 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).

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To mitigate risk, preventing contamination and testing for contaminants are complementary practices (13). Contamination risks exist in both aging facilities that are susceptible to mechanical failures, and new facilities that have operational unknowns. Between batches, there is a balance between decreased turn around time (thus maximizing production) and increased time for preventative maintenance (thus reducing failures) (21). Design, testing, and training each have an impact (22). Strategies for minimizing and investigating microbial (4, 14, 23, 24) and phage (24) contamination are available. DESIGN AND MAINTENANCE APPROACHES General fermentor design considerations Well-designed and maintained bioreactors and associated systems are important to achieving desired sterility and culture purity goals (2). Interestingly, many design features that typically are incorporated for sterility also are useful for containment (i.e., minimization of microorganism release) (25). For previously installed fermentors, the ability to cost-effectively 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, threepiece ball valves were found to be suitable and were implemented 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 hazardous energy control during maintenance (a United States Occupational Safety and Health Administration [OSHA] requirement) 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 surface 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 tradeoffs. Restricting automation and minimizing sequencing required operators in both the control room and field to observe the sterilization while making manual adjustments. There was greater manpower required and potentially higher variability, but reliability was increased if malfunctions unidentified by alarm conditions were determined immediately by personnel. General utility design considerations Both productcontact (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

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elevated above and placed upwind from fermentor exhausts and cooling tower mist. There was an estimated one-log reduction in live organism concentration with every 3 m of increased 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 elevated in temperature (26) but was cooled before reaching dryers and fermentors. In another facility’s installation, compressor after-coolers were removed entirely, and sparger air reached fermentors at 120°C. The facility’s monosodium glutamate fermentation was unaffected since heat was rapidly 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 compression to sterilize air even at low residence times of 15 s (21). The actual temperature at the compressor outlet depended on the ambient inlet air temperature, compressor load, and cooling tower water temperature to the compressor 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 chamber 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 dryers. Air then was filtered to remove desiccant before reaching fermentors. Liquid present in fermentor air filters was caused by (i) poor system design or maintenance, or (ii) excessive 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 fermentation 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 airlines with copper minimized leaks (particularly at fittings), maintaining reliable and adequate instrument air pressure. When the site instrument air supply was interrupted, a backup compressor and receiver tank prevented fermentor backpressure loss caused by automatic valves reverting to their failure states. General maintenance considerations A comprehensive preventative maintenance (PM) program, including product and non-product contact utilities, was implemented to minimize downtime. This program included a post-execution review of all associated documentation to ensure fol-

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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, especially those in constant steam service, (iv) annual testing of diaphragms on addition assemblies (valve arrays), both before and after repair, using pressurized air to detect leaks from valves submerged in water, (v) annual internal inspections for larger tanks to ensure bolts were present and tightened, 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 review 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 systems ensured changes were documented, communicated, and appropriately evaluated for potential effects on contamination as well as validation. Agitator The agitator seal was the main area of contamination 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 exposure 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 underwent 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 failure, 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 established 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 reduced 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 maintenance tests; higher vibration caused shaft runout and eventually seal leakage. However, proactive foot (steady) bearing replacement for 15,000 l fermentors was cost-effective every 1–2 years. Instrumentation Key instrumentation influencing contamination-free operation included fermentor back-pressure

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TABLE 1. History and type of probe failures (a) DO probes Total failures Year Failure/total 2004 5/199 2003 9/232 2002 21/271

% 2.5 3.9 8.5

Membrane 50 66.7 50

Failure type (%) Sensor Transmitter 40 0 27.8 0 15 5

Polarization 0 0 5

Cabling 10 0 17

Other 0 5.5 6

Repeats (%) 0 11.1 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 Year Failure/total 2004 1/199 2003 5/232 2002 6/271

% 0.5 2.2 2.2

Sensor 0 80.0 33.3

Transmitter 0 0 0

Failure type (%) Cabling Dehydration 0 0 0 0 16.7 16.7

and temperature instruments which were critical to sterilization reliability. Pressure gauges were verified against pressure transmitters at 1.1–1.3 kg/cm2, confirming their accuracy should transmitters become suspect. Resistance temperature detectors (RTDs) were characterized at three temperatures (0°C, 65°C, 130°C) using an oil bath (27). Cultivation (15°C to 45°C) and sterilization (0°C to 130°C) dual range RTDs were tested for agreement at growth temperatures (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 silicone-sealed on the product-contact side to prevent culture growth into the device (28). Differential pressure (DP) sensors in broth contact were examined carefully for leaks of sensing oil. For flange-mounted DP sensors, uniform torquing 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 conducted to ensure reliability. pH probe sensors were replaced every 6 months or 6 batches (whichever came first), dissolved 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 without bounce during the sterilization hold time, rose when the airflow started, and remained steady once fermentor temperature stabilized. After sterilization, pH probe perfor-

Other 100 20.0 33.3

Repeats (%) 0 20.0 0

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 detected, 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 associated 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 steamtraced manway installed on drug-oriented fermentors. The selected material, a high temperature peroxide-cured ethylene polypropylene diene monomer (EPDM) gasket, was required 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 inspection of filters being discarded ensured procedures were not imploding or otherwise damaging filters. The sparger air filter element was protected by an air prefilter and a steam filter (8). The steam supply valve to the sparger air filter was leak tested to prevent wetting and subsequent 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).

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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) clogging 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 pressure 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, including operational tolerances, ensured awareness of accuracy requirements. Establishment of piping installation preferences was particularly important since this facility was the only large-scale fermentation facility on site. For drug-oriented fermentors, these preferences included minimization of dead legs, installation of steam entries on the top of piping, 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 executed such that hazardous energy control locks were safely removed each day so equipment could be returned to service 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 impact. PROCEDURAL APPROACHES Pre-batch set up Fermentor integrity testing has mixed support in the literature. Some practitioners believed it unlikely 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 using 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 diaphragm valves on all inlet and outlet piping. Pre-batch integrity 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 bioconversion substrates were expensive, and (iii) using nutrient tanks idled for >2 months. Pre-batch set-up (as applicable to fermentor type) involved re-taping threaded national pipe thread (NPT) fittings, 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

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(IPA) in water to facilitate insertion and minimize damage. Personnel were trained to identify problems encountered inserting plugs or probes (as well as filters) rather than forcing 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 advance-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 without probes (since pH sensors showed degradation when exposed 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 leaving fermentors idle as a final cleaning step or after contamination with spore-forming bacteria (15). Longer advancesterilizations were undertaken at some facilities after repeated 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 obtaining 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-specific 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 internal 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 operation (e.g., elapsed time desiccant dryers were in service before triggering regeneration). Measures to undertake in the event of utility failures were documented. These procedures reduced the impact of product and non-product contact utility outages and promoted uniformity of response. After a utility failure, affected fermentors were evaluated to deter-

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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 executed for each fermentor after the annual maintenance shutdown to evaluate certain PM work such as instrument calibrations and control valve operation. If repair of an instrument or control valve was needed, post-repair testing was documented. Trends for multiple fermentors of similar scales helped quantify tolerable limits for accuracy and offsets, and a data history was developed. These batch sheets assisted in reducing the traditionally higher fall contamination rates (Fig. 1) and maintenance issues. Specifically, individual 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 reinstated. After shutdown, sparger air was blown through distribution 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 fermentor 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 accumulated hard water deposits. Vessel and media sterilization procedures To minimize bioburden, full cooling (6–8°C) was applied to drugoriented fermentors after batching but before sterilization. Batching and sterilization occurred in a timely manner; typically, 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 increases were not expected to be slow, however. Using backpressure control, sterilization target temperatures were achieved within 1°C. It was important to maintain 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 airflow was established, thus avoiding substantial backpressure drops. Raising backpressure to 1.5 kg/cm2 prior to introducing 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 application regimen. Preparation of inocula and mid-cycle additions Inoculation and mid-cycle additions are two process steps which potentially introduce open transfers either within biosafety cabinets and/or in the environment surrounding equipment. Minimization of risks associated with these steps primarily 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 particulate 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 sucking action likely disrupted laminar airflow patterns. Expo-

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sure plates also were not used since these plates added clutter, interfering if placed close to working areas where they were most relevant in detection. However, a segregated culture purity/sterility testing area was established to minimize cross-contamination with seed cultures. Although difficult to implement for a multi-purpose pilot plant, some production 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 Erlenmeyer flask stages (1×250 ml flask transferred to 3–6 ×2 l flasks), then pooled into one 4 l aspirator bottle per fermentor. Replacement of 2 l side-arm flasks with aspirator bottles containing bottom nipples (15) (i) avoided tipping of inoculum 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 presterilization of cotton (4), but this step was not adopted. For walk-in incubator rooms (which did not possess high efficiency 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 fermentor were prepared and tested for culture purity, eliminating 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 attachment of acid/base and antifoam containers was successful using large bore Chemap-style needles (Sartorius) with flaming of needles just prior to septum penetration to facilitate entry. Assembly preparation was aided using models and digital pictures, avoiding complicated written descriptions or diagrams. A few additional sterilized components were prepared 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-sterilize. Careful aliquotting procedures were developed to ensure 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-

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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 sanitized with dilute bleach before being refilled. All jugs were discarded annually. Pre-sterilization of nutrient feed solutions that were subsequently 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 autoclaving plastic jugs with numerous fittings. Filters were preferred on both the bag outlet and inlet (if not too expensive), just on the outlet if material transfer into the bag was presterilized, 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 transferred 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 backflow. The head pressure between the upstairs and downstairs 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 buildups 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 material from the source tank to the sewer; thus long transfers were avoided to conserve material. Another technique, backcooling 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 pressurizations were reversed to move from backward to forward flow; this step required valve integrity to minimize contamination risk. Careful attention to seed fermentor details (such as inoculation, sampling, and transfer line sterilization) was more important than the proximity of seed to production fermentors (4). During seed preparation the fewest possible transfers from seed vial to fermentor were desired (4). Post-inoculation mid-cycle additions (such as acid/base) were avoided in seed fermentations since the time for culture purity evaluation typically was short (1–2 d). Using one seed

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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 procedural 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 governed through unionized collective bargaining with unexpected turnover based on seniority and new personnel arriving 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 reference summary table (based on SOPs) comparing slight procedural 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 improve 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 desired) permitted a ready understanding of what the step accomplished. A balance was struck between avoiding significant procedural variation by individuals or shifts (4) and overly restricting when impact was negligible. Sufficient staff in both the microbiology support laboratory and the processing area was available so that work was efficient but not rushed. Scheduling at a reasonable pace relative to available personnel also allowed time for unexpected occurrences since the pilot plant environment was process development and not manufacturing-focused. A form for new processes, establishing to facility users what to consider in planning, was submitted well in advance of the expected run to permit discussion and mitigation of potential issues/risks. VALIDATION AND VERIFICATION Fermentor sterilization-in-place tests Two key concepts for sterilization were evaluated and tested (2). Thorough venting of air was confirmed by examining the temperature/pressure relationship (6). The use of superheated 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 fermentors, 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

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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 nutrients due to their lower vapor pressures (37). Higher temperatures also were used for low working volume sterilizations, especially of concentrated nutrients, to obtain adequate temperatures 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% cerelose 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 studies 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 mapping likely would have generated a more aggressive cycle. Fermentor sterility (media hold) tests SIP effectiveness for each drug-oriented vessel in this facility was confirmed 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 polypropylene 2000 (P2000; Dow, Freeport, TX, USA), adjusted to a pre-sterilization pH of 7.0. A low-end sterilization temperature 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 contaminated batch, and scheduling was arranged to accommodate 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 fermentor 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 increased (5.7 ± 7.3% from 1Q1990-3Q1997 vs 15.6± 6.1% from 4Q1997-3Q2004) as the facility’s annual contamination rate decreased (Fig. 2) since sterility batches were implemented 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

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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 temperature 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 sterilization 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 Erlenmeyer 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 transfer. Flask sterility tests assured cycle suitability for non-soluble 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 addition. Due to the lower heat transfer of plastics such as polypropylene versus glass (15), these bottles required notably longer autoclave times of 190 min for adequate sterilization. Filter and tubing manifold assemblies also underwent successful 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 examining 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

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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 highpressure 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, backpressure was removed slowly. This action avoided the controller 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 fermentors without all-welded internals. Finally, it permitted the cleaning agent to be maintained at its optimal temperature of 80°C (±10°C), under agitation at 75% of maximum speed, for the 1–3 d soaking duration. Shutting off all fermentor 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-pressurization or recirculation using a pump. If there was repeated contamination or uncontrolled foaming, more extensive 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 repeated contaminations (4), although the chemical interaction with stainless steel should be evaluated for the concentrations 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 bacterial/actinomyces, 2 yeast, and 1 E. coli), 3 biologics products (1 yeast, 1 E. coli, 1 animal cell) and 6 nutrients at several fermentor scales established the robustness of the facility’s cleaning strategy. Swabs revealed that Ingold-style ports had the highest total organic carbon (TOC) levels, possibly due to the 70% IPA applied to o-rings to facilitate insertion.

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MEDIUM SELECTION FOR DRUG-ORIENTED FERMENATIONS Medium components to avoid for drug-oriented fermentations due to contamination concerns were those ingredients 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 antifoams 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 vessel sidewalls where sterilization was less effective. Heat treatment of media at 80°C for 5–15 min during the sterilization heating phase aided dissolution of ingredients and enhanced germination of spores. An in-line mixer (homogenizer) disrupted clumps, ensuring 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 fermentor 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 settled solids. Direct charges of powders to fermentors without using a media mix tank were avoided, unless powders were slurried 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 sterilization of complex medium containing calcium carbonate required 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 reduced amount of any secondary carbon source, for the fermentor seed stage avoided excessively rich, solids-containing, 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

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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 macroscopic changes in on-line parameters (44), but were detected in off-line culture purity tests. In other cases, due to the time lag and discontinuous nature of culture purity testing, on-line variables showed contamination before off-line tests. Although contamination reduction efforts centered on improving equipment and sterile techniques, rapid contamination detection methods for inoculum and seed tanks still were required (35). Robust microbiological testing procedures have prevented use of contaminated inoculum, provided information on contaminations promptly, and pinpointed 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-consuming, classical microbiological methods (45). Over the years, culture purity testing regimens in this facility were refined and expanded to increase their sensitivity. Overly burdensome testing regimens were avoided. Incubation at both 24°C and 37°C detected both bacterial and fungal contaminants (33). Sampling for contaminants Sterility and culture purity analysis was conducted daily in this facility, and in other facilities once per shift (2), as well as after sterilization (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 (maintaining the surrounding area hot between samples), and it was cleaned regularly. One alternative was to submerge nipples 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. Pretransfer seed samples were taken about 8–16 h prior to production fermentor inoculation, and immediately sub-cultured. Pre-transfer samples were not taken prior to nutrient transfers since nutrient tank contamination rates were historically low at 0.7% (Table 2). Testing for contaminants Direct microscopic examination to identify contamination required distinguishing among culture, contaminant, and medium components (20). Thus, a sub-culturing enrichment technique was implemented at 37°C to supplement direct observation. Sub-culturing usually did not reflect the sample’s original popula-

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TABLE 2. Summary of contamination rates as a function of process type Process type Secondary metabolite Bioconversion

Constitutive/recombinant plasmid/protein Animal cell Nutrients (including water and defoamer) Total

Organism class (number of cultures) Filamentous bacterial/8 Fungal/19 Bacterial/5 Yeast/4 Filamentous bacterial/4 Fungal/3 E. coli/11 Yeast/5 Various/6 N/A Various

tion distribution (12) since it selectively enriched for contaminants. 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 cultures. Also in this facility, thioglycolate broth which detected 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; commercial preparations had screw caps which were not ergonomically attractive. To ensure adequate oxygen mass transfer, 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 examined visually for turbidity and unusual colony formation respectively 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 microscopically. 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, annotated 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 originated from pre-sterilization medium bioburden inactivated

Number of batches (contaminated/total) for process type 154/913 273/2483 9/79 8/365 5/49 16/70 19/249 2/214 5/31 6/749 531/5202

Contamination rate (%) Process type

Overall

16.9 11.0 11.4 2.2 10.2 22.9 7.6 0.93 16.1 0.8 N/A

3.0 5.2 0.17 0.15 0.096 0.31 0.36 0.038 0.096 0.12 10.2

by sterilization or live bioburden due to a contaminant. Reincubation of the TSB sub-culture for a few more hours, followed by re-examination and comparison to the prior field, permitted qualitative assessment of whether live rods increased in density. Fluorescence testing (LIVE/DEAD viability 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 similarly to contaminants. A contaminated fermentor was declared when two consecutive samples, taken at least 18 h apart to permit corrective action after investigation of the first contaminated sample, exhibited contamination. An unusual culture purity sample occurred when a sample was contaminated, but the subsequent 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 estimated 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) appeared 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 biochemical tests (47). Identification using fatty acid methyl ester (FAME) analysis generated the most likely genus and species 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-

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file matched the mean profile of a database organism was quantified by the similarity index: >0.5 indicated an excellent match, <0.3 indicated a species not likely in the database, and between > 0.3 and < 0.5 indicated a strain of the species that differed significantly from the database organism. 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 difficult to validate since bacteria show variability in growth and are not usually homogeneously distributed in a sample; thus, contaminants are unpredictable analytes compared to chemical compounds (49). Others even have used stereomicroscopes 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 substantial 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 perceived 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 detection devices currently have focused on sterility testing. Generally, they have not been appropriate for culture purity testing owing to difficulties distinguishing between desired and contaminating cultures. Interestingly once techniques were built into automated instruments, aspects of their methodology lost transparency (51), making it harder to determine applicability. A summary of available rapid methods for

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culture purity testing, along with benefits and limitations, is shown in Table 3. No one method emerges as widely accepted 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 potential problems was conducted in a non-judgmental manner to encourage openness (4). Anxiety associated with technique observations (particularly open handling steps), volunteering 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 impacted. 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 production 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 active, 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 disassembled. Immediately after removal, pH/DO probes were visually 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 conducted to detect external and internal leaks, including valve assemblies. Condensate traps were checked for pluggage. Internal tank surfaces were visually examined for cleanliness. 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 vessel full of water. About the only time the contamination investigation 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 removal. Assignment of most probable cause Common contamination causes were seed, raw materials, inadequate sterilization of equipment/air/media, inadequate procedures, insufficient training, procedures not followed, lack of routine PM, and bacteriophage (4). Other causes included construction materials, seals, valves, poor or overly complex design, operator errors, instrumentation (calibration, automated valve failure), sparger air, transfer/feed lines, contaminated in-

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TABLE 3. Summary of alternative rapid microbiological detection methods Principle

Method

Technique

Benefit/Limitation

Detection limit

Reference

Genetic markers/ polymerase chain reaction (PCR)

Battery of negative genetic markers- one set for each recombinant host

Growth on ribose minimal medium

Recombinant strain specific, sensitivity adjustable/Markers must be engineered into strain

1 in 106 to 1 in 1010 CFUs/ml of recombinant strains of same host cell type

54, McFarland and Swartz, Int. Pat. WO98/18955, 1998

Rapid detection of specific genes

Specific DNA segment amplification

Rapid/Considerable sample preparation effort

105 to 106 cells in food

55

Rapid (h or min)/Limited by primer specificity and completeness of primer library; gel identification of PCR product; inability to differentiate between live and dead organisms; designed for clinical and environmental samples

2× 107 prokaryotic cells in presence of eukaryotic (animal) cells

Webster, US Pat. 5348854, 1994

30 CFU/250 ml (0.12 CFU/ml) of lactic acid bacterium in beer

17, 44

Similarity of sample E. coli ribosome, hybridization with that RNA-specific, DNA of known contaminants probe

Light and radiometric

PCR via hybridization with DNA fragments of contaminants but not desired cultures

Amplification using universal primer pairs directed to different conserved parts of prokaryotic rDNA gene

PCR-Advanced nucleic acid analyzer (ANAA)

Real time PCR using TaqMan (homogeneous PCR using fluorescence resonance energy transfer) and spectrofluorometric thermal cycling

500 cells of Erwinia in medium

56, 57

Triangulation identification for the genetic evaluation of risks (TIGER)

Mass spectrometerderived base composition signatures from PCR amplification of highly conserved regions of microbial genome(s)

Single copy of contaminant genome specifically Bacillus anthracis in serum

58

Multi-parameter light scattering (MLS)

Interchanges of different optical polarizations resulting from polarized light scattering

1% B. thuringiensis in E. coli (1–5% depending on system)

59

Photon correlation spectroscopy

Dynamic light scattering to detect particle size

For 5% change in mean size: 1:71 ratio of E. coli to P. aeruginosa; 10:1 ratio of E. coli to S. cerevisiae

45

Scan RDI (Chemunex, Paris, France)

Laser scanning system; viable organisms converted fluoresceinbased substrate into fluorescent product

2 h detection/Selective lysis of mammalian cells required so they did not uptake fluorescent substrate

0.002 cells/ml in sterile medium (also used for animal cells)

11

Flow cytometry

Blood cell lysis, staining of bacteria with ethidium bromide followed by stained bacteria detection using flow cytometry

Rapid — <2 h, independent of contaminant growth/Electronic and hydro-dynamic “noise”, ability to differentiate “target” from interference, requires single cells to pass through system

10–100 CFU/ml, E. 60 coli in buffer and blood

Flow cytometry (Advanced Analytical Technologies, Ames, IA, USA)

Sample introduced to antibodies with fluorescent tags (syto 62 stain), counts by focusing on fluorescent events

Rapid; counts very low numbers/Counts everything — not just what grows — giving higher numbers

101–106 CFU/ml, Pseudomonas and Salmonella in media and in presence of animal cells

50, company literature

BACTEC bacterial growth promoter (Johnston Laboratories; Cockeysville, MD, USA)

Sealed vial of 14C inoculated with sample, 14 CO2 measured in ionization chamber

Faster than tubes and plates, established medical technique/Non-growth promoting medium or centrifugation minimized background readings from desired cultures with mixed results, radioactive substrate

Linear trade off between bacteria detection time and detection limit (100 CFU in 9 h, 105 CFUs in 6 h; sterile medium)

35

Real time, non-invasive, requires small liquid volumes/Low sensitivity unless contaminants larger than host organism-(less likely in commercial fermentations)

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TABLE 3. (continued) Principle

Method

Technique

Benefit/Limitation

Detection limit

Reference

Immunological

Binding with surface polysaccharides present in typical contaminants but not desired cultures

Fast immunogenic separation for enrichment followed by rapid enzyme immunoassay

Faster than growth methods/Only specific contaminating organisms can be targeted

> 5000 bacteria/ml (Salmonella) in 24 h/1 pathogen in 25 g of food

52

Chromatographic

Gas chromatography in conjunction with mass spectroscopy

Uses muramic acid and characteristic cellular fatty acids

Versatile, computerized evaluation/Profiles affected by growth phase and substrate; challenging to distinguish between contaminant and desired culture unless isolated

300 ng dry bacteria 500-fold bakers’ yeast; 0.25% (w/w) Staphylococcus and Bacillus, 0.5% Streptococcus, and 1 ppm — 1% Enterobacter cloacae — all in Leuconostoc

12, 48, 61

Different retention times of various size particles in empty column

260 µ id and 92 m length, particle detected by UV spectrophotometer

Independent of contaminant growth/Tested only with particles; size difference required; unable to distinguish live from dead

No actual cell systems tested

62

Differentiation of gross colony morphology and pigmentation

Varied agar media and incubation temperatures; growth differences

Cost-effective/Sensitivity; time required for growth; development required for substantially different desired cultures

< 100 CFU/ml in > 109 CFU/ml recombinant E. coli

Paul Duncan, personal communication

Morphological

On-line

< 100 CFU/ml in 5× 105 CFU/ml recombinant yeast 4–6 bacterial or mold CFUs/108 P. pastoris cells

13

Needs little prior process knowledge; algorithm adjustable to be more or less conservative/Challenging to define contamination alarm triggers; requires suitable on-line measurements

1% (w/w) E. coli in yeast culture

63

Contamination when real time process data varied ±25% from ideal data continuously for 30 min

Neural networks able to model non-linear processes/Training of mathematical model required

No direct testing done to detect contamination.

64

Electronic nose using array of conductive polymer sensors, responses analyzed using pattern recognition

“Real time” — does not require contaminant growth on plates/Sensors become oxidized or fouled; lot-to-lot sensor variability

No detection limit given for E. coli in M. carbonacea

65

Phenotype-based enrichment/selection

Varied media formulations based on carbon source as selective agent

Adaptive computer algorithm

On-line measurements of fermentation parameters

Artificial neural network model

eNose (model 4000; Osmetech, Roswell, GA, USA)

A recent summary of rapid microbiological methods also is available (66).

oculum, incorrect media sterilization, and inadequate procedures (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 facility, probable causes were grouped into four areas (Table 4): sterilization failure, mechanical (equipment problem), operational (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 improbable, 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 category 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 unknowns (55.3%), followed by contaminated transfers (25.1%) (Table 4). Substantial efforts then were implemented to improve thoroughness of post-contamination mechanical checkouts (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 highest percentage to equipment (54.6%) followed by unknown

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TABLE 4. Statistics for probable cause of contamination Probable cause (number/% of period’s total) Contaminated Failed Equipment Process transfer sterilization Overall 118.5/23.9 69/13.9 30/6.1 20.5/4.1 1Q1990-3Q1997 110.5/25.1 39/8.8 29/6.6 18.5/4.2 4Q1997-3Q2004 8/14.6 30/54.6 1/1.8 2/3.6 If evidence of more than one reason for failure suspected, each reason is counted on a fractional basis. Period

Unknown 258/52.0 244/55.3 14/25.5

TABLE 5. Statistics for type of contaminant Period

Cocci

Type of organism (number/% of period’s total) Gram-positive Gram-negative Fungal rod rod

Other

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 fractional 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 contaminant were useful for investigation (42). Since final identifications typically required a few weeks, quick communication 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 contact 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 sterilization of non-sterilized mid-cycle additions (42). Grampositive bacteria often entered from non-sterile air (8), owing to improper air filter installation, sterilization, or integrity (42). Gram-positive spore-formers possibly indicated incomplete sterilization owing to large medium particles or residual dried batch in vessel crevices (14). Yeasts sometimes 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). Multiple 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 contaminations in this facility where a definitive probable cause was ascertained; only some of these general trends were supported by the contaminant identification. In other facilities, the most frequent contaminants were spore-forming gram-positive or gram-negative rods; nonspore-forming bacteria usually were absent except when entering via jacket leaks (33). Fungal contaminations occurred

rarely (8). Fast-growing, spore-forming bacteria, such as B. subtilis, were the most common contaminants in Actinomycetes and fungal broths (6). These basic trends were consistent 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 contamination arose from transfer of undetected contaminated fermentor 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) increased during the period of 4Q1997-3Q2004; thus fewer batches were run annually. Monthly and annual data summaries 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 contamination rates were not readily obtainable from the database; thus it was roughly assumed that within a scale each fermentor 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

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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 introduction might have the greatest impact in further contamination rate reduction. SUMMARY AND IMPACT Contamination problems have delayed many fermentation plants from reaching their maximum production capacities (10). Due to a contamination wave, some facilities have discontinued the product associated with contamination and switched to another product (8). Some vessels only were used for specific products because of unresolved contamination issues that did not affect those particular products (53). Addressing pilot plant contamination was more challenging owing to the research rather than production environment. Since a wide variety of processes were undertaken, 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

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 deliveries and reliability of process development efforts. A proactive versus reactive approach directly affected productivity (avoiding transfer of contaminated batches), schedule (minimizing delays of subsequent batches while contaminations were investigated), manpower (reducing time devoted to investigation), and maintenance (reducing amount of disassembly and cleaning). Published benchmarks for contamination vary considerably. Despite problems in the pharmaceutical industry during 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

TABLE 6. Statistics for time of contamination detection Time of contamination detection (number/% of period’s total) AS AI ≤ 48 h >48–120 h >120 h Overall 39/7.9 79/15.9 204/41.1 95/19.2 80/16.1 1Q1990-3Q1997 32/7.2 74/16.7 176/39.8 85/19.2 75/16.9 4Q1997-3Q2004 7/12.7 5/9.1 28/50.9 10/18.2 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. Period

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exchangers of the continuous sterilization system (21). Constant attention to detail is required for sustained contamination reduction (53), and this commitment has been undertaken in this facility. The ultimate tool in controlling contamination is effective management and inter- and intradepartmental team building (including training, communication, and trust) among team members with varied functional backgrounds (2). Contamination awareness is maintained 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 acknowledgement goes to Dr. Peter Salmon who developed PARAFERM, a customized Paradox database from which much of the information for this paper was readily available.

REFERENCES 1. Chisti, Y.: Assure bioreactor sterility. Chem. Eng. Prog., 88, 80–85 (1992). 2. Perkowski, C. A.: Operational aspects of bioreactor contamination control. J. Parent. Sci. Technol., 44, 113–117 (1990). 3. Chaudhary, S. C. and Dodd, P. W.: The economic importance of sterility in industrial fermentation processes, p. 35–38. In Ghosh, P. (ed.), ICHEME- advances in biochemical engineering/biotechnology, vol. 51. Springer-Verlag, Berlin (1994). 4. Soderberg, A. C.: Fermentation design, p. 77–118. In Vogel, H. C. (ed.), Fermentation and biochemical engineering handbook. Noyes Publication, Park Ridge, NJ (1983). 5. Charton, R.: Simplifying sterile access in bioreactor operations. Am. Biotechnol. Lab., 8, 60 (1990). 6. Rhodes, A. and Fletcher, D. L.: Principles of industrial microbiology, p. 198–199. Pergamon Press, London (1966). 7. Saudek, E. C.: Symposium on industrial fermentations. Bacteriol. Rev., 20, 279–281 (1956). 8. Sharma, M. C. and Gurtu, A. K.: Asepsis in bioreactors. p. 1–27. In Neidleman, S. and Laskin, A. I. (ed.), Advances in applied microbiology. Academic Press, New York (1993). 9. Stockbridge, P. J., Hinge, R., Lawrence, A., Livingstone, A., Mercier, J. P., Pietrowski, R., and Wiersema, A.: Harvesting and disposition of foreign growth fermentors. Pharmeuropa, 13, 8–10 (2001). 10. Aiba, S., Humphrey, A. E., and Millis, N. F.: Biochemical engineering. Academic Press, New York (1965). 11. Onadipe, A. O.: Detection of microbial contamination in cell cultures using a laser scanning system. Biopharm, 14, 38–40 (2001). 12. Elmroth, I., Fox, A., Holst, O., and Larsson, L.: Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry. Biotechnol. Bioeng., 42, 421–429 (1993). 13. Plantz, B. A., Andersen, J., Smith, L. A., Meagher, M. M., and Schlegel, V. L.: Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths. J. Ind. Microbiol. Biotechnol., 30, 643–650 (2003). 14. Bader, F. G.: Sterilization: prevention of contamination. p. 345–362. In Demain, A. L. and Solomon, N. A. (ed.), Manual of industrial microbiology and biotechnology. American Society for Microbiology, Washington, D.C. (1986).

267

15. Kent, C. A., Dawson, M. K., Hearle, D. C., and Weale, D. J.: Sterilization, Ch. 8. In McNeil, B. and Harvey, L. M. (ed.), Fermentation: a practical approach. IRL Press, New York (1990). 16. Aunstrup, K., Andersen, O., Falch, E. A., and Nielsen, T. K.: Production of microbial enzymes, p. 281–309. In Peppler, H. J. (ed.), Microbial technology, vol. 1, 2nd ed. Academic Press, New York (1979). 17. Oakley-Gutowski, K. M., Hawthorne, D. B., and Kavanagh, T. E.: New developments in the brewing industry. Australas. Biotechnol., 1, 108–111 (1991). 18. Chang, I. S., Kim, B. H., and Shin, P. K.: Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation. Appl. Env. Microbiol., 63, 1–6 (1997). 19. Bayrock, D. P., Thomas, K. C., and Ingledew, W. M.: Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G. Appl. Microbiol. Biotechnol., 62, 498–502 (2003). 20. Bader, F. G., Boekeloo, M. K., Graham, H. E., and Cagle, J. W.: Sterilization of oils: data to support the use of a continuous point-of-use sterilizer. Biotechnol. Bioeng., 26, 848–856 (1984). 21. Bartholomew, W. H., Engstrom, D. E., Goodman, N. S., O’Toole, A. L., Shelton, J. L., and Tannen, L. P.: Reduction of contamination in an industrial fermentation plant. Biotechnol. Bioeng., 16, 1005–1013 (1974). 22. Spier, R.: Animal cells in culture: moving into the exponential phase. Trends Biotechnol., 6, 2–6 (1988). 23. Manfredini, R., Golzi Saporiti, L., and Cavallera, V.: Technological approach to industrial fermentation: limiting factors and practical solutions, p. 320–391. In Altanasowa, B. (ed.), Proceedings of the First International Conference on the Chemistry and Biotechnology of Biologically Active Natural Products. Bulgarian Academical Society, Sofia (1981). 24. Jones, D. T., Shirley, M., Wu, X., and Keis, S.: Bacteriophage infections in the industrial acetone butanol (AB) fermentation process. J. Mol. Microbiol. Biotechnol., 2, 21–26 (2000). 25. Brooks, C. H. and Russell, P. D.: A unified design approach to fermentor sterility and containment. Process Biochem., 21, 77–80 (1986). 26. Sylvester, J. G. and Coghill, R. D.: The penicillin fermentation, p. 219–263. In Underkofler, L. A. and Hickey, R. J. (ed.), Industrial fermentations. Chemical Publishing, New York (1954). 27. Junker, B., Brix, T., Lester, M., Kardos, P., Adamca, J., Lynch, J., Schmitt, J., and Salmon, P.: Design and installation of a next generation pilot scale fermentation system. Biotechnol. Prog., 19, 693–705 (2003). 28. Junker, B. H., Lynch, J., Leporati, J., Schmitt, J., Gieger, J., Garah, T., Stober, M., and Salmon, P.: Maintenance, equipment and operational improvements within an established fermentation pilot plant. Bioprocess Eng., 13, 279–287 (1995). 29. Boeck, L. D., Wetzel, R. W., Burt, S. C., Huber, F. M., Fowler, G. L., and Alford, J. S.: Sterilization of bioreactor media on the basis of computer-calculated thermal input designated as F0. J. Ind. Microbiol., 3, 305–310 (1988). 30. Orchard, T.: Containment of fermentor exhaust gases by filtration. Chem. Eng., 492, 17–20 (1991). 31. Reisman, H. B.: Economic analysis of fermentation processes. CRC Press, Boca Raton, Florida (1988). 32. Hasting, A.: Practical engineering for biochemical applications. World Biotech. Rep., 3500, 249–258 (1984). 33. Paladino, S., Ugolini, F., and Chain, E. B.: Fermentors of 90 and 300 l capacity for vortex and sparger aeration. Rendiconti Instituto de Sanita, 17, 88–120 (1954). 34. Junker, B., Seeley, A., Lester, M., Kovatch, M., Schmitt, J., Borysewicz, S., Lynch, J., Zhang, J., and Greasham, R.: Use of frozen bagged seed inoculum for secondary metabolite

268

35.

36.

37. 38. 39. 40. 41.

42.

43.

44.

45.

46. 47. 48.

49.

JUNKER ET AL.

and bioconversion processes at the pilot scale. Biotechnol. Bioeng., 79, 628–640 (2002). McLaughlin, J., Bruno, C. F., and Forrest, T.: A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations. Biotechnol. Bioeng., 25, 1229–1235 (1983). Stockbridge, P., Wilkinson, J., Servans, J.-P., Stewart, A., Wiersema, A., and Mitchell, R.: Validation of pure culture operations for large-scale fermentation systems. Pharmeuropa, 11, 565–570 (1999). Junker, B. H., Beshty, B., and Wilson, J.: Sterilization-inplace of concentrated nutrient solutions. Biotechnol. Bioeng., 62, 501–508 (1999). Junker, B.: Technical evaluation of the potential for streamlining of equipment validation for fermentation applications. Biotechnol. Bioeng., 74, 49–61 (2001). Singh, V., Hensler, W., and Fuchs, R.: Optimization of batch fermentor sterilization. Biotechnol. Bioeng., 33, 584– 591 (1989). Chisti, Y. and Moo-Young, M.: Clean-in-place systems for industrial bioreactors: design, validation and operation. J. Ind. Microbiol., 13, 201–207 (1994). Corbett, K.: Design, preparation and sterilization of fermentation media, p. 127–139. In Moo-Young, M. (ed.), Comprehensive biotechnology, vol. 1. Pergamon Press, Oxford (1985). Junker, B.: Good manufacturing practice (GMP) and good industrial large scale practices (GLSP), p. 1355–1367. In Flickinger, M. C. and Drew, S. W. (ed.), Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation. John Wiley and Sons, New York (1999). Junker, B. H., Hesse, M., Burgess, B., Masurekar, P., Connors, N., and Seeley, A.: Early phase process scale up challenges for fungal and filamentous bacterial cultures. Appl. Biochem. Biotechnol., 119, 241–277 (2004). Spaepen, M., Angulo, A. F., Marynen, P., and Cassiman, J. J.: Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction. FEMS Microbiol. Lett., 99, 89–94 (1992). Jepras, R. I., Perkins, E. A., Rarity, J., Carr, R. J. G., Clarke, D. J., and Atkinson, T.: Application of photon correlation spectroscopy as a technique for detecting culture contamination. Biotechnol. Bioeng., 38, 929–940 (1991). Reisman, M. E.: Sterility now. Pharm. Formul. Qual., January/February, 22–26 (1998). Akers, J. E.: Biotechnology product validation: isolator technology applications. Pharm. Technol., 19, 28–40 (1995). Elmroth, I., Valeur, A., Odham, G., and Larsson, L.: Detection of microbial contamination in fermentation processes: mass spectrometric determination of gram-negative bacteria in Leuconostoc mesenteroides cultures. Biotechnol. Bioeng., 35, 787–792 (1990). Curlee, J.: Validating microbiological methods: seeking guidance in a sea of uncertainty. Pharm. Cosmet. Qual.,

J. BIOSCI. BIOENG.,

July/August, 19–21 (1998). 50. Lewandoski, N.: Is there time for rapid micro? Pharm. Formul. Qual., April/May, 17–22 (2001). 51. Flickinger, B.: Making dollars and sense with rapid microbiology. Pharm. Formul. Qual., April/May, 22–28 (2000). 52. Van Beurden, R. and Mackintosh, R.: New developments in rapid microbiology using immunoassays. Food Agric. Immunol., 6, 209–214 (1994). 53. Perkowski, C. A., Daransky, G. R., and Williams, J.: Detection of microscopic leaks in fermentor cooling coils. Biotechnol. Bioeng., 26, 857–859 (1984). 54. Swartz, J. R. and McFarland, N.: Genetic approaches to the detection of contaminants in Escherichia coli fermentations. Biotechnol. Prog., 14, 88–91 (1998). 55. Hill, W. E. and Wekell, M. M.: Some practical applications of gene probes and PCR to food safety, p. 537–541. In Ladisch, M. R. and Bose, A. (ed.), Harnessing biotechnology for the 21st century. American Chemical Society, Washington, D.C. (1992). 56. Belgrader, P., Benett, W., Hadley, D., Richards, J., Stratton, P., Mariella, R., and Milanovich, F.: PCR detection of bacteria in seven minutes. Science, 284, 449–454 (1999). 57. Jimenez, L.: Light up the life in microbial contamination. Pharm. Formul. Qual., August/September, 54–55 (2001). 58. Hofstadler, S. A., Sampath, R., Blyn, L. B., Eshoo, M. W., Hall, T. A., Jiang, Y., Drader, J. J., Hannis, J. C., SannesLowery, K. A., Cummins, L. L., and 22 other authors: TIGER: the universal biosensor. Int. J. Mass Spectrom., 242, 23–41 (2005). 59. Fraatz, R. J., Prakash, G., and Allen, F. S.: A polarization sensitive light scattering system for the characterization of bacteria. Am. Biotechnol. Lab., 6, 24–28 (1988). 60. Mansour, J. D., Robson, J. A., Arndt, C. W., and Schulte, T. H.: Detection of Escherichia coli in blood using flow cytometry. Cytometry, 6, 186–190 (1985). 61. Elmroth, I., Sundin, P., Valeur, A., Larsson, L., and Odham, G.: Evaluation of chromatographic methods for the detection of bacterial contamination in biotechnological processes. J. Microbiol. Methods, 15, 215–228 (1992). 62. Shiragami, N.: A simple and rapid method for the detection of contaminations in a bioreactor. Bioprocess Eng., 5, 115– 117 (1990). 63. Chattaway, T. and Stephanopoulos, G. N.: An adaptive state estimator for detecting contaminants in bioreactors. Biotechnol. Bioeng., 34, 647–659 (1989). 64. Bhowmik, U. K., Saha, G., Barua, A., and Sinha, S.: Online detection of contamination in a bioprocess using artificial neural networks. Chem. Eng. Technol., 23, 543–549 (2000). 65. Namdev, P. K., Alroy, Y., and Singh, V.: Sniffing out trouble: use of an electronic nose in bioprocesses. Biotechnol. Prog., 14, 75–78 (1998). 66. Moldenhauer, J.: Rapid microbiological methods and the PAT initiative. Biopharm. Int., 18, 31–46 (2005).