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THOMAS J. KEHOE Beckman Instruments, Inc., Fullerton, Calif.
Status of Continuous pH Measurement within Pharmaceutical Fermentation Processes
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potential usefulness of continuous p H measurement within fermentation processes has been recognized by the industry for some time. Many pharmaceutical companies have attempted continuous p H measurement with various degrees of success. An informal survey was made 2 years ago to determine the full scope of existing problems, successes to date, and future requirements of the industry. Because of the recent accomplishments and increased interest on the part of the pharmaceutical industry in continuous p H measurement, it was believed advisable to review the present status of application. This survey was aimed at summarizing the opinions, requirements, and results obtained by the industry. All known organizations, directly or indirectly related to fermentation processes, were sent a detailed questionnaire with an explanatory cover letter, in an effort to gather as complete a cross section as possible. The response was most gratifying. Results of Survey
The 42 companies and organizations surveyed included 23 believed to be actively engaged in fermentation processes, and 19 organizations indirectly concerned. Included in the latter group were commercial and governmental research organizations and consultants, with several fermentation equipment manufacturers known to be close to this subject.
Degree of Response
ClassiGcation Actively engaged Indirectly concerned Total
Response t o Questionnaire ReNo plied reply Total 21 2 23
'9
9, 30
12
42
General Status of Application. As expected, the group classified as indirectly concerned unanimously stated that
it had no direct experience beyond the laboratory bench scale. Of the 21 that replied that were considered actively engaged in fermentation processes, only four had never performed experimental work on continuous p H measurement within fermentors. The remaining 17 have spent from 1 to over 5 years investigating continuous p H measurement within fermentors. The average experience of all in the group is 3.5 years. Nine of the 17 claimed some degree of success in employing the measurement, while eight stated that their efforts were unsuccessful. Only one company stated that it has saccessfully employed continuous p H in full-scale production size fermentors. Causes of Failure. Those that could not report successful continuous measurement were asked to list the cause(s) of failure.
Causes of Failure No. of Difficulty Responses Glass electrode failure 7 Reference electrode failure 4 Leakage of electrode housing 3 Contamination due to electrode breakage 2 2 Coating of electrode Sample recirculation problems 1
I t is evident that the basic complaint stems from glass electrode failure. I n view of the various successful operations described, wherein standard glass electrodes have withstood repeated steam sterilizations, a point of confusion might exist in respect to the proper placement of the prime cause of failure. The rather broad and sometimes confusing choice of available glass electrodes may have resulted in use of electrodes not capable of high temperature applications. Expected electrode life is a n arbitrary estimation. A 2-week life in one plant might be considered satisfactory, while another plant might expect 12-month life, despite the rather severe application involved.
Physical Methods of Measuring pH. The industry was asked to state its choice in respect to the actual mechanical method of measuring pH at the fermentors.
Methods of Sampling
Sampling Method
Direct insertion of electrodes Recirculating external sample stream Continuous sample bleed t o waste
Number Responding Pilot Proplant duction scale scale 14
11
2
4
3
5
Potential Contamination D u e to Reference Electrode Salt Bridge. A point of frequent concern regards the potential hazard of exposing the fermentor contents to the potassium chloride filling solution from the reference electrode. The contents of a reservoirtype electrode mounted externally through the fermentor, or in a recirculating sample stream, may not be fully sterilized by the steam, as only the lower portion may receive the full thermal benefit of the steam. A variety of opinions prevail on this subject. The majority believe that addition of one of various chemical sterilizing agents tQthe potassium chloride solution adequately solves this problem. Four disagreed with this conclusion. The amount of filling solution that can enter a fermentor (excluding mechanical breakage) is dependent upon the style of liquid junction employed. One interesting possibility exists in the use of the palladium wire type of junction. By the nature of its construction, it is conceivable that this type of junction would serve as a filter or barrier, preventing the passage of harmful organisms present in the potassium chloride solution. Several plants were outspoken in approving the use of chemical sterilizing agents, with the one qualification that it must not be present in excess of the concentration necessary to sterilize the VOL. 50,
NO. 9
SEPTEMBFR 1958
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potassium chloride solution. Thus, a complete breakage of the electrode, permitting entry of the entire contents of the electrode into the fermentor, still will not sterilize the batch. Principal Difficulties Encountered by Users. Those surveyed were asked what basic obstacles still exist that should be corrected. The responses varied considerably, except for consist-
Principal Difficulties Encountered No. of Specific Problem Responses Short l i e of electrodes 28 Inadequate mechanical seal around electrodes 2 Reference electrode filling solution 4 Subjecting complete electrode assembly to steam sterilization 1 Mechanical breakage of electrodes within fermentor 1 Need for longer electrodes for small-scale fermentors 1 Need for very small electrodes for laboratory scale fermentors 1 Reducing electrode coating prob1 lem _. 39 Total
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ent complaint regarding short electrode life. No one stated that reference electrode fouling was a problem. On the other hand, only four replied that they employ air pressure to the electrode to oppose steam sterilization pressures, while nine stated that they have never used opposing pressure. Entry of foreign solution into the reference electrode often causes ultimate measurement failure. I t is possible that some lack of success in this application was primarily due to failure of the reference electrode, although other reasons were attributed. Conclusion
The response to this survey, with observation of the activities of various groups, indicates that considerable progress has been made by the industry in employing continuous pH to fermentation processes. O n the other hand, considerable effort must still be spent in improving techniques and p H components and in settling questions such as the acceptability of sterilized potassium chloride solution, before this application can be universally adopted.
This review only reports the findings of the survey. Other recent articles have described highly successful pH systems where the difficulties mentioned above have been overcome. The practical experience gained and techniques employed by such successful installations should be thoroughly studied to gain the benefit of their approach. Because of the widely different policies that exist between different organizations, it would be most difficult to describe a universally reliable pH system acceptable to all. O n the other hand, each successful installation can offer methods or procedures that can be incorporated into various approaches in the establishment of a specific type of pH system for other installations. Acknowledgment
Sincere appreciation is expressed to the many individuals throughout this industry who contributed their opinions, comments, and suggestions by means of the questionnaire and by personal contact. Their interest and cooperation will greatly facilitate progress in improving and simplifying this application.
FRANK W. DENISON, Jr., IRVING C. WEST, MERLIN H. PETERSON, and JOHN C. SYLVESTER Abbott Laboratories, North Chicago, 111.
Large-Scale Fermentations
A PracticaI System for pH Control This electrode assembly for controlling pH in large-scale fermentations can withstand steam sterilization
OPTIMUM
pH RANGES have been determined for most industrially significant fermentations (7-4). Chemical buffers such as calcium carbonate, phosphates, and citrates, used to maintain such ranges, have narrow limits of application. For submerged pure-culture fermentations, nutrients are frequently added which on utilization produce the desired pH. However, adding acid or base during fermentation is more desirable, because nutrients can then be selected for maximum product yield. Control on a continuous automatic basis requires that the electrodes be in
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constant contact with the beer. Chemical sterilization (3, 4) prior to aseptic insertion of the electrodes into the fermentor is considered too hazardous because of the danger of contaminating high value production batches. Attempts have been made to sterilize electrodes in place with steam (3, 6, 7) but repeated failure of the glass electrode makes this procedure impractical for routine operation. This report describes a method for controlling pH of submerged fermentations, using steam sterilization of the electrodes.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Design and Operation
The 50-gallon stainless steel fermentor with a 30-gallon operating volume is similar to larger scale equipment, but has the flexibility essential for this type of study. In the bell-shaped p H electrode chamber (Figure l), similar to one previously described (6), were mounted at hightemperature glass electrode (Beckman No. 8990-90) a temperature compensator, and a reference electrode. The chamber was placed in the fermentor and lead wires were brought out through
