Simple Strategies to Improve Bioprocess Pure Culture Processing

Reprinted from PHARMACEUTICAL ENGINEERING® The Official Magazine of ISPE May/June 2010, Vol. 30 No. 3 Pure Culture Bioprocessing www.ISPE.org ©Copyright ISPE 2010 This article presents Simple Strategies to Improve strategies to improve Bioprocess Pure Culture Processing bioreactions by reducing contaminations by Michael Hines, Chris Holmes, and Ryan Schad by adventitious agents. ioreaction and fermentation processes of traditional bioprocess reactors and fermenta- of all scales – from 100,000 liter vessels tion processes. They are targeted squarely for to small development reactors – require the practitioner in their simplicity, and based pure culture for their successful, pro- on many years of managing pure culture opera- Bductive operation. In this article the term “pure tions at many scales. The focus of this article culture” and “pure culture capability” will be is on preventing bacterial contaminations in used instead of “sterile” or “sterility assurance” traditional fermentation systems; much of the to acknowledge that bioreactions are, by nature, material applies to protection of any bioreactor the process of growing large populations of systems from any adventitious agent. It is up to helpful microorganisms or cells as opposed to you to understand your system and process and the more unwanted varieties. Contamination to appropriately apply the principles outlined in by adventitious agents costs time, money, and order to meet the requirements of your process lost productivity; moreover, contaminants and and business. Higher potential risk from longer their sources can be very difficult to locate and growth times, longer inoculum trains, and lack eliminate. of selective agents (e.g., antibiotics) can be offset Many elements of pure culture bioprocess through the use of disposables, newer equip- design and operation are straightforward, often ment, and more stringent environmental and even a matter of common sense. Most modern raw material controls. facilities have pure culture capability built into The focus of this work is divided into four their design, including latest technology and basic sections: 1. design aspects for sterile op- control systems, and correct operating proce- erations; 2. common contamination root causes; dures to ensure sterility is achieved and pure 3. troubleshooting FG events; and 4. applying culture maintained. However, these controls will rigorous microbiology to better understand become less effective over time, due to deterio- and improve pure culture. Additionally, several ration, obsolescence, loss of experience, process examples and case studies are included to il- changes, and personnel turnover. Sometimes, lustrate and emphasize concepts. many years of solid performance can lead to a false sense of stability, which leads to com- Facility Design and placence (or a lack of attention to pure culture Sterilization Best Practices capability requirements) inevitably followed by The scope of this article is for scientists and one or more Foreign Growth (FG) episodes. engineers supporting existing bioreactor Ultimately, a process with robust pure culture processes. However, a short survey of current capability is the goal of both technical sup- sterile design best practices will be helpful to port staff as well as management. It is clearly improve or troubleshoot your axenic process. beneficial to proactively manage pure culture It is fundamental to realize that prevention of design, capability, and practices before problems FG is the most important factor to long-term occur rather than create unintentional sterility successful pure culture performance. experts out of your support staff as they struggle with root cause investigations instead of more Sterilize in Place (SIP) System Design productive efforts. Considerations for Sterile Operations Contained below are a number of strategies, Design for optimal sterilization is covered in illustrative case studies, and techniques to help many texts and articles.1,2 Main points are es- maintain or improve pure culture capabilities sentially as follows: MAY/JUNE 2010 PHARMACEUTICAL ENGINEERING 1 Pure Culture Bioprocessing Pure Culture Bioprocessing • Using steam, quick heat up of all points in the sterile bound- Air Removal – air must be completely displaced by saturated valves, care must be taken to control steam temperature, flow, Setup: Microbial fermentor experienced sporadic foreign growths, especially ary to 121.1°C (121.1°C is the United States Pharmacopeia steam for sterilization to be effective. Air must be either pulled common during periods of high tank volume or “foam outs.” Extensive and differential pressure across the diaphragms in order to (USP) standard sterilization temperature with a minimum by vacuum or displaced effectively by the steam itself. Typi- investigation into source of contamination did not reveal any sterile boundary prolong service. Use of condensate seals as opposed to steam moist heat sterilization requirement of 15 minutes.)3 cally, air is discharged through a sterilizing filter. flaws except a very minor one: the insulation around the top of the Vapor seals also can prolong valve elastomer life. Liquid Separator (VLS) had been removed for repair work some time before, • free drainage of condensate and was not replaced. • easy displacement of air Cold Spots – temperature measurement must include the Trade-Off between In-Line and Off-Line Monitoring Devices Resolution: Replacing the insulation decreased the frequency of foreign • replace collapsing steam with sterile air (collapsing steam coldest spot in the system to ensure that all points are held growth events, but did not eliminate them. Further investigation revealed – in some cases, the number of required in-line monitoring creates vacuum, which must be avoided at all costs) above sterilization temperatures. Redundant temperature that the VLS had a stationary Clean-In-Place (CIP) spray ring with spray devices can be reduced by using external lab sampling or in- measurement is essential to verify sterilization temperatures holes drilled on the side of the ring, not the bottom, so that the cleaning direct relationships between key operating parameters. The solution was ejected horizontally outward from the spray ring instead of These, and other factors to consider are described below: are maintained. vertically downward. appropriate number and location of analytical instruments and in-process checks must be reconciled against: 1. capital Consequently, the Sterile Boundary – process piping connected to sterilized Equipment Drains – all areas in process equipment must spray ring was not free and operating cost constraints; and 2. pure culture concerns, equipment must be sterilized up to and through the closest be totally drainable. Ideally, all equipment surfaces should draining and always due to the fact that an increasing number of instrument ports valve which isolates the sterile from the non-sterile system. drain toward one common bottom outlet (each drain point had a heel of water raises FG risk to the bioreactor. lying stagnant within, Another way to isolate the sterile system is through an ap- is a potential cold spot). A steam trap should be installed to where environmental propriate 0.2 micron-rated sterilizing grade filter. Other sterile remove condensate in this outlet. bacteria could gain a Dead Legs – the piping configuration of the sterile system is boundaries include vessel walls themselves, mechanical seals foothold between runs. one of the most critical attributes that contributes to main- Sterilization process in (subject to pressure gradient), feed nozzles, internal cooling Setup: During an investigation of a recurring Foreign Growth (FG) the upper portion of the taining a system free of FG. Avoiding so called “dead legs” is coils, rupture discs, sterilizing filters, steam traps, exhaust contamination series on a large microbial fermentor, experienced operators VLS was adequate for crucial. Basically, a dead leg is defined as a one-way system, noted that the equipment SIP dynamics had shifted in the past few weeks. lines, and o-rings (elastomers) on instrument ports. See a surface sterilization, typically on the end or a branch of a piping distribution sys- Specifically, the system was reaching correct sterilization temperatures, but was not as more detailed discussion of the sterile boundary, located in but was at a higher pressure in order to meet the required temperature. effective in sterilizing tem, which results in a process hold-up area that is difficult the next section on contamination root causes. Additionally, FG contamination events were intermittent and also seemed to the heel of water to clean and sterilize. The ASME BPE standard suggests that correlate with high level or “foam out” events in the system. when the insulation Figure 2. Schematic sketch of an internal bioprocessing systems, such as fermentation, along with other was removed and was CIP spray ring with no drain hole. Disposables – disposable bioreactors and attachments are Resolution: Detailed investigation found that a routine automation change bioprocesses, should be designed with a target L/D ratio of 2:1. on some unrelated control parameters inadvertently and subtly altered occasionally ineffective gaining in popularity because they can reduce risk of cross- sterilization logic. The SIP logic change resulted in a minor delay in the even with the insulation in place. When the process broth or foam refluxed to L is defined as the length of the dead leg extension

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