On-line analysis and control of production of antibiotics

Analytica Chimica Acta, 213 (1988) l-9 Elsevier Science Publishers B.V., Amsterdam -

ON-LINE ANALYSIS ANTIBIOTICS

Printed in The Netherlands

AND CONTROL

OF PRODUCTION

OF

K. SCHUGERL Institut fiir Techniwhe Chemie, Universitiit Hannover, Callinstr. 3., D-3000 Hannover 1 (Federal Republic of Germany) (Received 27th February 1988)

SUMMARY The prerequisites for the utility of on-line techniques for monitoring and control of biotechnological production processes are discussed. Aseptic filters, air-segmented continuous-flow automatic analyzers, flow-injection analyzers and on-line HPLC systems are discussed. These were used for the process analysis and control of penicillin V and cephalosporin C production in stirredtank and airlift-tower loop reactors. The results of on-line and off-line analyses are compared, as are different analytical methods. The suitability of these techniques for process control is demonstrated in several examples.

For the biosynthesis of antibiotics, coordinated cooperation of several enzymes is necessary. The activity of a particular enzyme depends upon the concentration of several components of the medium (substrates, products, inductors, repressors, inhibitors and activators). Some of these are known, while others have not yet been discovered. In order to achieve the full biological potential of the cells, the optimal production conditions must be maintained at least with regard to the most important key parameters. The first step towards this goal is identification of the key components. This is usually done by optimization of the medium. However, this method does not give information on the regulation of biosynthesis. Acquisition of this information requires special runs combined with on-line analysis of the medium and cellular components. In addition, knowledge of the dynamic behavior of the microorganisms is required for process control and for coping with operational disturbances. Though the fluid dynamics of the broth has a considerable influence on the bioreactor performance and the production process, the present paper considers only the measurement of the concentratton of broth components and their use in process control. First, the on-line methods which are used for process analysis and control of antibiotic production in the author’s laboratory are considered. 0003-2670/88/$03.50

0 1988 Elsevier Science Publishers B.V.

2 ON-LINE ANALYSIS

An extremely important prerequisite for on-line techniques is long-term reliability. All the components used should be able to operate automatically through the night and during the weekend without supervision. For continuous sampling, it is necessary to use aseptic filters which can be steam-sterilized at 121’ C and can operate without back-flushing for many hundreds of hours. Gas analyzers (infrared CO, and paramagnetic 0, analyzers, and mass spectrometers) and also the pH and pop electrodes have been found to be highly reliable. Experience with broth component analyzers has shown that only online high-performance liquid chromatography (HPLC) and some flow-injection analyzers meet this reliability requirement. The weak points of air-segmented continuous-flow automatic analyzers are the peristaltic pumps, which can break down, and the tubing and the coils, which may become blocked by precipitated proteins or by micro-organisms which can grow in the nutrientrich permeating flow. In spite of these disadvantages, such analyzers are very popular in biotechnology laboratories. Flow-injection analyzers are flushed with a continuous flow of substrate-free buffer which is interrupted only by a short pulse of the medium; thus, no extensive protein precipitation and micro-organism growth can occur. They also have shorter response times than the airsegmented analyzers, because, unlike air-segmented analyzers, they work in the kinetic range without an equilibrium being established. Samples for on-line HPLC analysis must be deproteinated. It is necessary to prepare the eluents with twice-distilled water (or commercially available pH 7 phosphate buffer) and chromatographic-grade methanol; these must be filtered through an ultrafiltration membrane and ultrasonically degassed. In addition, the eluent can be degassed during the fermentation with helium. The use of an HPLC precolumn is also recommended. For automatic operation, computer-controlled procedures for flushing, calibration and blank correction as well as data acquisition, evaluation and control are necessary. Figure 1A shows a shows a discus-shaped filter (with a free filtration area of 13.5 cm2, a dead volume of 1.53 ml, and a response time of 3.5 min); Fig. 1B shows a porous-plate filter (with a free filtration area of 47.5 paws le-

(A)

115 mm*

rigs-

Fig. 1. (A) Discus-shaped

filter [ 11. (B) Filter with porous-plate support [ 21.

plate

3

cm2, a dead volume of 2.5 ml and a response time of 9 min). The porous-plate filter is used in stirred-tank reactors and the discus-shaped filter is used in an airlift-tower loop reactor; both filters utilize polysulfone membranes (with a molecular weight cut-off of 100 000). They are positioned in the lower section of the reactors in a well-aerated region. By means of the hydrostatic pressure in the reactors alone, permeation rates of 0.5-1.0 ml min-’ can be maintained, as long as the broth does not contain lard oil. When in situ sterilization is used by condensing 121 ‘C steam into the reactor, the discus-shaped filter was found to be more reliable. This filter had an operational time of about 1000 h without membrane back-flush [ 31. The air-segmented flow analysis system (Skalar Analytika, Breda, NL) used in this laboratory consists of peristaltic pumps, a reaction section and a detector, with automatic operation and control of calibrations, blank determinations, specific determinations, cleaning and regeneration. The system is operated with a microprocessor (ct-68000, GWK) connected via standard RS232 asynchronous serial lines to a PDP-11/23 computer. The control of data acquisition, processing, and fermentaton is done by a computer automation system for fermentation apparatus (CASFA; Institut fiir Technische Chemie, Universitat Hannover ). The various continuous automatic analyzer channels are shown in Table 1. For flow injection analysis, several systems have been used: a YSI 23A carbohydrate analyzer (Yellow Springs Instruments, Ohio) which was rebuilt for on-line analysis (Mischke E. G.) [4]; and MOR (Institut fiir Technische Chemie, TCI) which was operated with buffer recirculation [ 7,8], an enzyme thermistor [9,10] and a biocalorimeter [ 111. Information on these flow-injection systems is summarized in Table 2. TABLE 1 Air-segmented automatic analyzer channels [ 3-61 Species

Method

Detector

Phosphate Sulfate Ammonium Urea Penicillin DOCb

Ammonium molybdate Ba-methylthymol blue/ion-exchanger NaOH/ETDA, membrane Diacetylmonoxime, thiosemicarbazid Hydroxylammoniumchloride/nickel chloride K-peroxodisulfate/UV, membrane

Reducing sugar Methionine Cephalosporins

p-Hydroxybenzoic acid hydrazide Na nitroprusside Absorbance of cepham chromophore’

Photometer Photometer ISE” Photometer Photometer Micro-CO2 gas sensor Photometer Photometer Photometer

“Ion-selective electrode (Orion). bDissolved organic carbon. “With blank after enzymatic cleavage of p-la&am ring.

4 TABLE 2 Flow-injection

systems

[ 7-111

Species

Immob. enzyme/system

Detector

Glucose

Glucose oxidase/membrane YSI 23A, Mischke EG. Lactate oxidase/membrane YSI 23A, Mischke E.G. NaOH/ETDA/membrane MOR, TCI Glucose oxidase/VA-Epoxy BIOSYNTH MOR, TCI Lactate oxidase/VA-Epoxy BIOSYNTH MOR, TCI fi-Galactosidase/glucose oxidase/VA-epoxy BIOSYNTH/ MOR, TCI cu-Glucosidase/glucose oxidase VA-epoxy BIOSYNTH/MOR, TCI Urease/VA-Epoxy BIOSYNTH MOR, TCI Glucose oxidase/catalase Penicillinase/CPG-10 and/or Eupergit C Penicillin G-amidase/CPG-10 and/or Eupergit C Penicillinase/Eupergit C Penicillin-G-amidase/Eupergit C Penicillin-G-amidase/Eupergit C Penicillin-G-amidase/Eupergit C Glucose oxidase/catalase Eupergit C Urea (in solution) Penicillin G (in solution)

H,O, electrode

Lactate Ammonia Glucose Lactate Lactose’ Maltose’ Urea Glucose Penicillin G Penicillin G Penicillin V Penicillin V Ampicillin Carbenicillin Glucose Urease Penicillin G amidase

“Orion. bEppendorf,

Hamburg.

‘Not yet optimized.

dEnzyme thermistor,

H,Oz electrode NH,-gas

sensor”

Oz electrodeb O2 electrodeb Op electrodeb Oz electrodeb NH,-gas sensor” Enz. therm.d Enz. therm. Enz. therm. Enz. therm Enz. therm. Enz. therm. Enz. therm. Biocalorimeter Enz. therm. Enz. therm

University

of Lund.

TABLE 3 Broth components determined by on-line HPLC [ 1,12,13] with tetrabutylammonium hydrogensulfate/methanol or (for reversed-phase operation) phosphate buffer/methanol as eluent Penicillin

Cephalosporin

V broth

Phenoxyacetic acid K-penicillin V p-Hydroxypenicillin Penicilloic acid Penilloic acid

V

C broth

Methionine Deacetyl-cephalosporin C 2-Hydroxy-4-methylmercaptobutyric Deacetoxy-cephalosporin C Penicillin N Cephalosporin C

acid

The /I-lactam and side-chain precursors and the decomposition product in the broth were determinedvia on-line HPLC [ 1,12,13]. The broth components which were determined on-line are given in Table 3. The dissolved oxygen

5

concentration and the concentrations of O2 and CO2 in the gas phase were also measured, and thus the volumetric mass-transfer coefficients (k,a) and respiratory quotients (RQ) could be determined on-line. OFF-LINE ANALYSIS

All components which are determined by on-line analysis, were also determined by off-line procedures. The amino acids and oligopeptides in the broth and cells were determined by HPLC methods, as were the intracellular concentrations of j?-lactam precursors. For calculation of the yield coefficients for penicillin V production with Penicillium chrysogenum, the cell mass was determined gravimetrically with the sampling system described by Kiinig [ 141, after separation of the solid substrate components [ 151. A pellet suspension was maintained in the airlifttower loop reactor. The size distribution of the pellets was measured by sieving the pellets in water. Because the Cephulosporium acremonium mold adhered to the solid peanut flour, so that it was not possible to separate them, the RNA content of the sediment was determined and the cell mass was calculated from it by using the method of Kiienzi [ 161. Carbon, hydrogen and nitrogen were determined with a CHN-Rapid analyzer (Heraeus). COMPARISON

OF THE DIFFERENT

There is good agreement Fig. 2, off-line and on-line C sodium salt (CPC ) and production of cephalosporin from determinations made

METHODS

between the on-line and off-line measurements. In HPLC data for the concentrations of cephalosporin deacetyl cephalosporin C are compared during the C. In Fig. 3, glucose concentrations are compared off-line and on-line with YSI 23A, off-line with the

Fig. 2. Comparison of on-line and off-line determinations. (A) Na-cephalosporin C (CPC) concentrations determined by off-line ( A ) and on-line HPLC during CPC production [6]; ( x ) CPC determined fresh off-line. (B ) Deacetylcephalosporin C (DAC ) concentrations determined by offline (A ) and on-line HPLC during production of CPC [ 121; the arrows indicate changes in the feed.

6

Fig. 3. Comparison of glucose concentrations determined off-line and on-line: (A) with the YSI 23A during production of CPC [4] (0, off-line data); (B) wih the biocalorimekr on-line and by MOR off-line ( A ) during the production of penicillin V [ 111.

0

0 .

OO

1

54

I

I

108

162

I

216

1

-

270 Q

Lo o

fermentation time [hl Fig. 4. Comparison of lactate concentrations measured off-line with the MOR ( x ) and YSI 23A (a) systems and on-line (enzyme reactor) during the production of penicillin V [ 31.

Fig. 5. Comparison of glucose concentrations and the YSI 23A ( 0 ) during the production

fermentation time [h] measured off-line with an enzyme thermistor of penicillin V [ 111.

(A )

7

MOR, and on-line with the biocalorimeter, during the production of CPC and penicillin V. In Fig. 4, lactate concentrations are compared which were determined off-line (MOR, YSI 23A) and on-line (enzyme reactor). In Fig. 5, glucose concentrations determined off-line with the YSI 23A compared with those determined off-line with an enzyme thermistor during the production of penicillin V. ON-LINE ANALYSIS

AND CONTROL

Penicillin Vproduction The production of penicillin V is strongly influenced by the concentrations of dissolved oxygen, glucose/lactose, nitrogen source and side-chain precursor (phenoxyacetic acid). At low glucose/lactose concentrations, the growth rate and the productivity are low. When the glucose concentration is too high, repression occurs and the productivity diminishes. Therefore, the glucose concentration must be maintained at the optimal level in the broth. This was achieved by on-line determination of glucose and fed batch operation. The same statements are true for the ammonium (N-source) concentration. Figure 6 demonstrates how the ammonium concentration was kept at its optimum level by fed batch operation. At very low concentrations of the precursor, the productivity is low, but when the precursor concentration is too high, the mold oxidizes the phenoxyacetic acid at the p-position. This yields the p-hydroxy-

+ 80

120

160

200

o

fermentation time [h]

Fig. 6. Ammonium and penicillin V concentrations penicillin V [ 3 ] ; ( 0 ) ammonium feed rate.

determined on-line during the production of

8

0

40

200

240

ferl~ntati~t? time1E] Fig. 7. Precursor (phenoxyacetic acid, POAA) and K-penicillin V (KPV) concentrations mined with on-line HPLC during the production [ 11.

deter-

penicillin V. To avoid the formation of this by-product and to keep the productivity high, an optimal precursor concentration must be maintained. This was achieved by on-line determination of precursor concentration and fed batch operation (Fig. 7). Energy is wasted when an unnecessarily high dissolved oxygen concentration is maintained, but when the concentration of dissolved oxygen is too low, transfer limitation occurs, the cells are damaged, and the productivity drops to zero. There is an optimum concentration at l&20% of the saturation value. However, if antifoam agent, lard oil or precursor is fed to the broth, the specific interfacial area is suddenly reduced and the dissolved oxygen concentration drops to zero. To avoid this situation, the dissolved oxygen concentration is usually increased to a level considerably above the optimum, and energy is wasted. However, by using three parallel connected oxygen electrodes and parameter-adaptive control, the pOZ in the broth can be maintained at its optimal value. Cephalosporin production The glucose and methionine concentrations must be maintained at their optimum values to attain the maximum productivity and to avoid a low yield of methionine, which is converted partly to 2-hydroxy-4-methylmercaptobutyric acid if its concentration is high. Therefore, the concentrations of reducing sugars and methionine were determined on-line during the production phase (Fig. 8)) and adjusted as required. When peanut flour was used, the phosphate concentration remained constant ( x 10 mg 1-l) during the entire fermentation time. Also, the ammonium concentration was kept nearly constant (0.40.5 g 1-l) by pH control. As soon as the glucose concentration was increased to release the glucose repression, the cephalosporin C production stopped and the cell growth in-

9

0

m

40

60

80

c

IIll

IO0

120

I.0

160

0

c

Ihl

Fig. 8. Monitoring during the production of CPC [4]: (A) concentration of reduction sugars determined on-line by the photometric method (Table 1); concentration of methionine measured by on-line HPLC.

creased. When the dissolved oxygen concentration was reduced below its critical value, the cephalosporin C production stopped, because the enzyme for the ring extension was blocked, and penicillin N was enriched in the broth. By means of on-line analysis and control the productivities of these processes were increased considerably The author gratefully acknowledges the financial support of the Ministry of Resarch and Technology of FRG and the Volkswagen Foundation, and the support of Hoechst AG, Frankfurt and Ciba-Geigy, Basle. He also thanks Drs. Th. Bayer, Th. Herold, J. Moller, J. Niehoff and A. Sauerbrei, likewise T. Dullau, K. Holzhauer-Rieger, B. Reinhardt and W. Zhou for their excellent cooperation.

REFERENCES

10 11 12 13 14 15 16

J. Mijller, Doctoral Dissertation, University of Hannover, 1987. T. Lorenz, Doctoral Dissertation, University of Hannover, 1985. J. Niehoff, Doctoral Dissertation, University of Hannover, 1987. T. Bayer, Doctoral Dissertation, University of Hannover, 1987. J. Niehoff, J. Mijller, R. Hiddessen and K. Schiigerl, Anal. Chim. Acta, 190 (1986) 205. T. Bayer, T. Herold, R. Hiddessen and K. Schtigerl, Anal. Chim. Acta, 190 (1986) 213. T. Finkeldey, Diploma Thesis, University of Hannover, 1987. B. Reinhardt, Diploma Thesis, University of Hannover, 1987. B. Danielsson, B. Mattiasson, R. Karlsson and F. Winquist, Biotechnol. Bioeng., 21 (1979) 1749. B. Danielsson, F.C. Mandelius, F. Winauist, B. Mattiasson and K. Mosbach, in M. MooYoung (Ed.), Advances in Biotechnology, Vol. 1, Pergamon, Toronto, 1981, ppl4d45. A. Sauerbrei, Doctoral Dissertation, University of Hannover, 1987. K. Holzhauer, Diploma Thesis, University of Hannover, 1987. J. Miiller, R. Hiddessen, J. Niehoff and K. Schtigerl, Anal. Chim. Acta, 190 (1986 ) 195. B. KBnig, Doctoral Disssertation, University of Hannover, 1980. J. Wittler, Doctoral Dissertation, University of Hannover, 1983. M.T. Ktienzi, Biotechnol. Lett., 1 (1979) 127