- Email: [email protected]
Immunological on-line detection of specific proteins during fermentation processes
Analytica Chimica Actu, 249 (1991) 113-122 Elsevier Science Publishers B.V., Amsterdam
113
Immunological on-line detection of specific proteins during fermentation processes Ruth Freitag *J , Christel Fenge, Thomas Scheper and Karl Schiigerl Institut
ftir Technische Chemie, Universitiit Hannover, Caliinstr. 3, D-3000 Hannover I (Germany)
Andreas Spreinat and Garo Antranikian Instiiur ftir Mikrobiologie,
Georg-August-Universittit
Giittingen, Grisebachstr. 8, D-3400 Giittingen (Germany)
Elisabeth Fraune B. Braun Melsungen AG, Schwarzenberger (Received
Weg 73, D-3508 Melsungen (Germany)
20th November
1990)
Abstract A system for real-time monitoring of specific proteins in fermentation processes is introduced. For measurements the turbidity caused by aggregates formed between the proteins to be detected and their antibodies is measured photometrically at 340 nm. The flow-injection analysis principle was used to automate the assay fully, including calibration and washing steps. The assay was intended for on-line product monitoring. It was therefore optimized to antibodies (mab) cover concentration ranges of l-1000 mg l- ‘. The analyser was used to measure monoclonal produced in fermentations of mouse-mouse hybridoma cells and to quantify pullulanase isoenzymes produced in a fermentation of Clostridium thermosuifurogenes. The fermentations took between 240 and 450 h, including extended phases of steady-state production. During that time, the long-term stability of the analyser system was excellent. A relative standard deviation of less than 2% was calculated for the data. A conventional ELISA served as a reference assay in the mab measurement. The enzymatic activity found in comparable off-line samples was used to correlate the on-line pullulanase measurements. Correlation coefficients between 0.94 and 0.99 were found between the on-line assay and the reference assays. Keywords:
Flow system;
Turbidimetry;
Bioprocess
monitoring;
The rapid development of biotechnological production methods over the last decade makes large-scale industrial production of biologically active substances conceivable. Close monitoring of the process is not only mandatory for substances to be used for therapeutic applications in humans, but also a necessary part of process optimization. Control and regulation of parameters such as tem-
’ Present address: Department of Chemical Engineering, University, New Haven, CT 06511, U.S.A.
0003-2670/91/$03.50
0 1991 - Elsevier
Process
analysis;
Proteins
perature, oxygen content and pH can be taken for granted in most modern fermentation processes. More and more analysers are being introduced for the monitoring of low-molecular-weight nutrients and metabolites [1,2]. Many of these analysers are fully automated and can be interfaced to a computer-based process control system. The substance produced, however, will often be a protein or other molecule of high molecular weight. Little has been done to permit the real-time determination of the concentration and stability of these products.
Yale
Science Publishers
Fermentation;
B.V.
114
The discriminatory power of antibodies has made immunoassay the most suitable candidate for this type of detection. Antibodies can be raised against virtually all proteins. The reaction between the antibody and the protein (antigen) is highly specific and sample preparation therefore becomes less complex. The assay described was designed to follow product formation rather than the build-up of some impurity. Concentrations of up to 1 g l- ’ had to be expected. In order to keep the system as simple as possible a turbidimetric immunoassay [3,4] with a detection range of 1 X 10P4-1 g 1-l was chosen, rather than an assay with a lower detection limit connected with a sophisticated appliance for sample dilution. The principle of flow-injection analysis (FIA) is the most important method for the automation of wet chemical analysis [5,6]. A given reaction does not have to reach equilibrium, as sample dispersion in an FIA system is highly reproducible. This is especially important for the automation of antibody-antigen reactions, which are based on the encounter of certain areas of large molecules [7]. The aim in this work was to establish a method for the on-line detection of specific proteins and to prove its potential for long-term monitoring during real fermentation processes. The analyser was successfully employed to monitor the production of monoclonal antibodies by hybridoma cells during two fermentations. A slightly modified assay was used to follow the production of a thermostable enzyme by bacteria.
EXPERIMENTAL
Flow-injection analyser for on-line detection of specific proteins A merging-zones stopped-flow FIA system as shown in Fig. 1 was assembled for assay automation. The different elements were activated by a set of interacting time-switch relays. The two loops for sample and sample blank were continuously flushed with fermenter supematant. Complete replacement of residual buffer was assumed after flushing the injector with ten loop volumes of sample. The reagent loop was filled discontinuously after displacement of residual buffer by an
R. FREITAG
ET AL.
1I
L-!-l
t
buffer
Fig. 1. Schematic diagram of the flow-injection analyser used to automate the turbidimetric immunoassay. The different elements of the system are controlled by a set of time-switch relays. The data are transferred to a computer for processing.
air bubble. Antibody consumption was thus reduced. Sample and reagent were injected into separate buffer streams. After merging, the flow was interrupted for a certain incubation time to allow the aggregates to form. After incubation the turbidity of the reagent mixture was measured. In the meantime a sample blank was established in the other section of the FIA manifold. Buffer vessels and reaction coils were thermostated at 37 o C. A detection wavelength of 340 nm (Scalar 6300 filter photometer) was chosen. This wavelength is far enough from the protein absorption maximum of 280 nm to ensure a low background noise, while the signal intensity is still high compared with readings taken at longer wavelengths. The data were transfered to a modified Atari computer and converted into antigen concentrations by means of a calibration graph [8]. Both peak height and area could be used for this purpose. The system was recalibrated at regular intervals. Washing steps, e.g., of the sampling line, could be included when necessary.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
115
PROCESSES
probe, the harvest pump drew the fermenter contents through a static filtration unit equipped with a hydrophilic microporous tubular membrane. As proteins and cells were not able to traverse the membrane, they were retained in the fermenter. Analysis. Off-line samples were taken daily and aliquots stored at -20” C for further analysis after clearing by centrifugation. Cell densities were established with a haemocytometer, using the Trypan Blue dye exclusion test to differentiate between viable and non-viable cells [lo]. The amino acid content of the fermenter supematant was measured by reversed-phase liquid chromatography after precolumn derivatization with o-phthaldialdehyde. Overall protein contents were determined according to Bradford [ll]. An automated analyser was connected to the CSTBR for glucose and lactate measurements (YSI 2000; Yellow Springs Instruments). A BIOPEM filtration module (B. Braum Diessel) was used to obtain a continuous sample stream [12]. A height-adjustable stirrer was placed above the filtration membrane to prevent filter-cake for-
High cell density perfusion culture of IgG-producing hybridoma cells Procedure. Mouse-mouse hybridoma cells (cell
line DB9G8, PTCC HB 124 [9]) were propagated in Dulbecco’s modified Eagle’s medium (DME) supplemented with controlled process serum replacement (CPSR) 4 (both from Sigma). The cells produce monoclonal antibodies (IgG,,, K-light chain) against human insulin. A 1.6-1 continuously stirred tank bioreactor (CSTBR) equipped with control mechanisms for temperature, pH and dissolved oxygen (Biostat MC; B. Braun Diessel) was used. The experimental set-up is shown in Fig. 2. The dissolved oxygen was controlled at 20% air saturation and the pH at 7.2. A temperature of 37O C was maintained throughout. Fresh culture medium was continuously pumped into the CSTBR during the perfusion phase of the experiment. Dilution rates were increased from l.Od-’ to 1.52d-’ during the first and from 0.8d-’ to l.ld-’ during the second fermentation according to the nutritional needs of the increasing cell population. Activated by a level
YSI
option
2000
II
!!
2
I lmmuno
1
FIA
feed
TFU
BIOPEM
lmmuno n FIA BIOPEM YSI
I > harvest
bioreactor
1 2000
8
option J
1
.
pump
Fig. 2. Perfusion bioreactor for the production of monoclonal antibodies by hybridoma cells. A Yellow Springs YSI 2000 analyser for glucose and lactate measurement and a flow-injection analyser for IgG measurements are connected to the fermenter via two sampling modules. For sampling the BIOPEM module is used either by itself or connected with a tangential filtration unit (TFU) as sterile barrier.
116
R. FREITAG
filtrate
outlet
inlet
Fig. 3. Tangential filtration with a 0.22~pm membrane the BIOPEM if necessary.
unit (TFU). The unit was equipped and used as a sterile barrier after
mation. If neccessary, a tangential filtration unit (TFU) (Fig. 3) was connected in series with the BIOPEM. The units were fitted with 0.22~pm PVDF (polyvinylidendifluoride) membranes (Durapore type; Millipore) or modified PTFE membranes of different pore sizes. Turbidimetric immunoassay (TIA) for measurements of monoclonal mouse antibody (mab). Anti-
mouse IgG (Sigma M-7023, developed in rabbit, titre 1.8 mg ml-‘) was used as a reagent. A 0.01 mol 1-i sodium phosphate buffer (pH 7.2) con-
ET AL.
taining 30 g 1-l PEG 6000 and 4.5 g 1-i sodium chloride (all from Fluka) was used throughout. The flow-rates of the buffer and sample streams were adjusted to 1 ml mm’. Reagent was pumped at 0.1 ml min- i. Injection loops of 0.05 ml were used in all instances. The detection range was l-1000 mg 1-i mab. A purified mouse IgG (Sigma I-5381) was used at several dilutions to obtain the calibration graph given in Fig. 4. An incubation period of 90 s was necessary, adding to the 150 s per analysis. During the experiments the system was recalibrated using standard IgG concentrations of 1,100 and 1000 mg 1-l. Frequent washing with Decon 90 (Zinser Analytik) was necessary to prevent protein build-up in the system. The analyser was automatically activated three times a day for a triple measurement. A conventional sandwich enzyme-linked immunosorbent assay (ELISA) was used as a reference assay for the TIA. Affinity-purified goat anti-mouse IgG was used both as a coating and as a detection anti-
.
360
720
Fig. 4. Calibration graph for the turbidimetric assay of monoclonal mouse antibodies. The signal intensities are given in ODs, units, since the term optical density can be used regardless of the physical phenomenon causing the observed decrease in light intensity. In our case this decrease is due to the scattering of the incoming light by the immunocomplexes, rather than to e.g. an absorbance of the light.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
body. For the latter purpose the antibody was conjugated to alkaline phosphatase. All reagents for the ELISA were obtained from Dianova (Hamburg), except the IgG standard (Sigma I5381) used for calibration. Continous culture of Clostridwm thermosulfurogenes Procedure. The production of three thermostable pullulanase isoenzymes by a Clostridium EMl, species (Clostridium thermosulfurogenes
Deutsche Sammlung ftir Microorganismen 3896) was monitored. A starch-limited medium [13] was utilized for cultivation. A 2-1 BCC fermenter (Schtitt, Gottingen) equipped with in-line sensors for temperature and pH and a deep tube for off-line sampling was used. The experiment was done under anaerobic conditions. The temperature was kept at 60” C throughout and the pH was regulated at 6.0 during the continuous culture by adding appropriate amounts of sterile 2 mol 1-l potassium hydroxide solution. A cross-flow hollow-fibre module (KF 200 010 S ME; Microgon) was connected to the
00
240
480 Pullulanase
720 [U/l]
117
PROCESSES
bioreactor to obtain a representative sample stream for the on-line analyser. Analysrs. Off-line samples were taken every 2 h during the batch phase and three times daily during the continuous culture. The samples were stored at 0” C for further analysis. Cell densities were determined by measuring the turbidity of the sample at 578 nm against cell-free fermenter supernatant. Pullulanase concentrations in the fermenter and in the on-line sample stream were measured according to Bergmeyer et al. [14]. Turbidlmetric Immunoassay (TIA) for pullulanase. The assay is to a large extent similar to
that described above for mouse IgG. Here 0.05 mol 1-l sodium phosphate buffer (pH 7.4) was used. The PEG content was increased to 40 g l- ’ while the sodium chloride concentration remained at 4.5 g 1-l. A longer incubation period of 120 s proved to be beneficial. Anti-pullulanase antibodies were obtained by inoculating a rabbit with purified pullulunase produced by C. thermosulfurogenes. The antibodies reacted in an Ouchterlony double diffusion test [15] with all pullulanase isoenzymes. The final antiserum was di-
960
1200
Fig. 5 Cabbratlon graph for the turbldlmetnc puIIulanase assay See also remark m legend to Rg 4
118
R. FREITAG
luted 1: 40 using the reaction buffer without PEG as dilutant. A detection range of 10-1000 U 1-l was established using purified pullulanase as a standard. The dose-response curve is given in Fig. 5. The system ‘was recalibrated using pullulanase concentrations of 0, 10, 100 and 1000 U 1-l.
RESULTS AND DISCUSSION
Fermentations of hybridoma cells Production with mammalian cells constitutes a as these major challenge in biotechnology, organisms require a highly controlled environment. Growth conditions and production rates of the hybridoma cell line HB 124 have been investigated in the Melsungen laboratory for some time [16]. The growth kinetics for the fermentations performed with on-line product monitoring did not differ significantly from those observed before. During the initial batch phase the cell density increased exponentially from 0.14 X lo6 to 1 x lo6 viable cells ml-‘. Exponential growth was maintained during the first 2 days of the perfusion
ET AL.
culture and the specific growth rate calculated was 1.2 d-l. Subsequently the culture entered a stationary phase. In both experiments final cell densities of ca. 2 x 10’ viable cells ml-’ were reached. During the steady phase, glucose and lactate levels could be stabilized at 2 and 1 g l-‘, respectively. Amino acid concentrations were held at constant levels of ca. 50% of the original medium concentrations. Glutamine and methionine concentrations were below 10%. As glutamine is important for cell metabolism [17], growth and production might be restricted under these conditions. Supplementing the regular medium with glutamine should therefore be beneficial. In a perfusion culture proteins are retained within the bioreactor [18,19]. The advantages of high cell and product concentrations are self-evident. Overall costs are lowered as the amount of serum or serum substitute used to enrich the culture medium can be gradually reduced during the fermentation. In this instance the CPSR 4 content was lowered from 4% at the beginning to 1.1% towards the end of the cultures. This also improves the protein to product ratio. The resulting
Time [h]
Fig. 6. Protein and monoclonal antibody concentrations during the original fermentation of HB 124 cells. The protein concentration of the fermenter supematant was measured according to Bradford [ll]; the antibody concentration was measured in centrifuged off-line samples using a sandwich ELISA for mouse IgG.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
mab and overall protein concentrations for the original fermentation can be taken from Fig. 6. IgG levels of 3% were reached at the end of the first and 4.68,at the end of the second fermentation. Although the fermentation process itself and the established analysis connected with it did not cause any difficulty, the acquisition of a representative sample was found to be a major obstacle m the on-line determination of proteins. For reasons of asepsis, the membrane dividing the nonsterile analyser and the culture vessel should not have pore sizes larger than 0.22 pm (sterile barrier). Several set-ups were studied during the original fermentation. The BIOPEM was fitted with a 0.22-, a 10 or a 5-pm membrane. If necessary, the TFA equipped with a 0.22~pm membrane was connected to it. Protein and mab concentrations were measured on both sides of all filtration membranes. Within 70-100 h the permeability of the BIOPEM membrane decreased to ca. 50%. The permeability for mab was usually better (ca. 80%). The decrease in permeability was found to be independent of the pore size; a 5-pm membrane did not perform better than a 0.22~pm membrane in the BIOPEM. Unfortunately, the stirrer in the BIOPEM did not prevent clogging and concentration polarization. A 0.22~pm membrane utilized as
PROCESSES
119
an additional sterile barrier after the primary sampling unit, however, demonstrated no further tendency to retain proteins. This emphazises the importance of cell debris and large membrane proteins for filter fouling. Although the large dead volume (200 ml) of the BIOPEM constituted another problem, the module was utilized again during another fermentation. The unit was fitted with a 0.22~pm membrane and exchanged daily. Adequate sampling thus became possible. The construction of a sampling device suitable for proteins, however, remains a challange to engineering. Product monitoring was archived during both experiments, despite the difficulties caused by the sampling procedure. The results are depicted in Fig. 7. During the original culture the mab concentrations did not increase as usual. This was to be expected, as the concentrations in the BIOPEM sample stream were not directely correlated with the mab level reached in the fermenter. Consequently, the behaviour became more normal during the second fermentation. The sharp decrease in mab concentration after 320 h is caused by dilution of the fermenter contents with fresh culture medium. A relative standard deviation of less than 2% was calculated for the measurements. If the product concentrations established with
140
200
300
400
500
Time [h] fig % C’oncentratlons he
DB9G8
ATCC
of monocional- anthodies HB 124 0 = Expenment I,
measured- turbldimetncally expenment II.
A =
diumg
two perfuslon
cultures
of hylindbma
cellq celI_
R FREITAG
120 250
++
+ ++ + +
+
50 4 *+ +* L.9 0
50
I. 100
I. 150
200
Mouse IgG [mgll], TIA Fig. 8. Correlation between mouse IgG measurements obtamed with the turbldlmetnc immunoassay and urlth the ELISA reference assay for samples with ldentlcal background The fermenter supernatant was clanfled by fdtratlon and samples were analysed m an off-hne mode with both methods A correlation coefflclent of 0 98 was calculated.
prolonged storage. with the former.
Process control
ET AL
is only possible
Fermentation of Clostrthum thermosulfurogenes Protein production by rmcroorganisms such as bacteria is more important than production by mammalian cells, as these organisms are less demanding and easier to propagate. The production of a Clostndzum species was followed by a specific immunoassay. The bacteria excrete three isoenzymes of a thermostable pullulanase [20]. Pullulanases are debranching enzymes capable of hydrolysing a-1,6-glycosidic linkages in lngh-molecular-weight polymers of D-glucose. The thermostability of the pullulanases produced by C. thermosulfurogenes in connection with the pullulytic ability makes these enzymes extremely interesting to the food industry.
0
CI
Pullulanase [U/I], Fermentationbroth centrituged 80 160 320 240 I
I
I
400
I
4
4
9
the TIA and the ELBA are compared, good agreement is found for concentrations below 15 coefficient of 0.94 was mg 1-l. A correlation calculated. For higher concentrations the correlation became worse. During the onginal experiment a correlation coefficient of 0.83 was found. The correlation improved for the second fermentation, a value of 0.96 being obtained. Generally, concentrations ascertained with the ELBA are lower than those produced by the TIA. A certain amount of protein deterioratron must be assumed during sample storage prior to the ELISA measurements. When the TIA was employed m an off-line mode to analyse samples after storage, the correlatton with the ELISA measurement was better, as shown in Fig. 8 (correlation coefficient 0.98). Although the problem of obtaining a representative sample remains unsolved, the amount of time and the manual labour involved in product monitoring were considerably reduced. Mab concentrations measured in fresh fermenter supernatants should be superior to those obtained after
4 <\
E
16I -
z t I= 27
36
45 0
80
160
240
Puklanase
[U/l], TIA
320
400
Fig 9. Values for pullulanase concentrations measured turbtdlmetncally and with the enzymatic actlvlty reference assay dunng the first 20 h of C thermosulfurogenes fermentation Fermenter supematant samples were centnfuged and ahquots used for both types of measurements.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
Pullulanase monitoring proved to be much simpler than IgG monitoring. During the batch culture the automatic analyser was not activated. Instead, aliqupts of samples taken off-line were cleared by centrifugation and injected manually into the TIA system. Correlation with the activity measurements was good, as can be deduced from Fig. 9. A correlation coefficient of 0.99 was calculated. In the continuous phase a single cross-flow hollow-fiber filtration module was employed for the entire 244-h fermentation. During that time no significant difference was observed between the pullulytic activity of samples taken before and after the module. The continous culture was started after 20 h, when the pH of the fermenter content had decreased to 4.01. A dilution rate of 0.075 h-’ was adjusted initially. After 80 h (4.5 residence times), an equilibrium between bacteria growth and production rates and the dilution rate was reached. After four more residence times the dilution rate was raised to 0.1 h-i. The cell density did not stabilize again, but a new plateau for pullulanase was archived. Pullulanase production is induced by a high-
.
800
W _ F 2,
600
PROCESSES
121
molecular-weight polymer of D-glucose as main source of carbohydrate in the culture medium. If this source is replaced with glucose itself, pullulanase production ceases. Such an exchange from a starch to a glucose basis took place after 200 h. After 244 h the pullulanase concentration had decreased to zero and the experiment was terminated. Cell growth and pullulanase production as measured by the TIA and the activity assay are shown in Fig. 10. A correlation coefficient of 0.94 was calculated for the two assays. The relative standard deviation for the TIA was again below 2%. The detection limit of 10 U 1-l constituted no problem as this concentration was surpassed after 10 h of fermentation. The longterm stability of the system was excellent. Repeated recalibrations showed no major shift in the calibration graph. During the initial batch culture, the higher sensitivity of the enzyme activity assay makes that method preferable, especially as process control is not possible at this point. Once a stable steady state has been reached the continuous production of pullulanase can continue for several months. In
z
W 2 f
ti
400
i z 200
0
0
50
150
100
200
TIME [h] Fig. 10. Cell growth activity assay.
and pullulanase
concentration
measured
on-line
with the turbidimetric
assay
and off-line
with the enzyme
122
R. FREITAG
this phase, when the detection limit is no problem, the immunoassay will simplify process maintenance and safety supervision.
Conclusions The industrial-scale production of biologically active compounds demands the development of new techniques for their on-line assessment. Experimental results indicate that the FIA immunoassay system introduced here allows on-line monitoring of product formation in continuous long-term cultivation processes. The inclusion of high-molecular-weight substances in the group of process control parameters thus becomes feasible. This work was supported by a DECHEMA grant to R. Freitag, grant No. 0318815A from the Bundesministerium ftir Forschung and Technologie, and by the BMFT within the Schwerpunkt: Grundlagen der Bioprozesstechnik.
REFERENCES 1 M. Cam, M. Gisin and C. Thommen, Biotechnol. Bioeng., 28 (1989) 423. 2 M. Mason, in C.P. Hollenberg and H. Sahm (Eds.), Biosensors and Environmental Biotechnology, Biotec, Vol. 2, Gustav Fischer, Stuttgart, New York, 1988.
ET AL.
3 G.A. Hudson, R.F. Ritchie and J.E. Haddow, Clin. Chem., 27 (1981) 1838. 4 P.J. Worsfold and A. Hughes, Analyst, 109 (1984) 339. 5 J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 114 (1980) 19. 6 W.E. van der Linden, Anal. Chim. Acta, 179 (1986) 91. 7 W.R. Griswold, J. Immunoassay, 1 (1987) 145. 8 R. Freitag, Th. Scheper and K. Schligerl, Anal. Chem., submitted for publication. 9 J. Schroer, T. Bender, R.J. Feldmann and K.J. Kim, Eur. J. Immunol., 13 (1983) 693. 10 W. Halle, Zell- und Gewebeziichtung bei Tieren, Gustav Fischer, Stuttgart, 1976. 11 M.M. Bradford, Anal. B&hem., 72 (1976) 248. 12 K.-H. Kroner, W. Stach and W. Kuhlmann, Chemie-Technik, 15 (9) (1986) 74. 13 G. Antranikian, P. Zablowski and G. Gottschalk, Appl. Microbial. Biotechnol., 27 (1987) 75. 14 H.U. Bergmeyer, K. Gawehn and M. Grabe, in H.U. Bergmeyer (Ed.), Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 3. Auflage, 1974. 15 6. Ouchterlony, Acta Pathol. Microbial. Stand., 26 (1949) 507. 16 E. Fraune, Ch. Fenae and W. Kuhlmann. 8th International Biotechnology Symposium, Paris, 1988, Abstract 115. 17 L.J. Reitzer, B.M. Wice and D. Kenell, J. Biol. Chem., 254 (1979) 2669. 18 Y. Takazawa, M. Tokashiki, K. Hamamoto and H. Murakami, Cytotechnology, 1 (1988) 171. 5 (1989) 48. 19 D. Siewert, BioEngineering, C. Herzberg and G. Gottschalk, Appl. 20 G. Antranikian, Environ. Microbial., 53 (1987) 1668.
113
Immunological on-line detection of specific proteins during fermentation processes Ruth Freitag *J , Christel Fenge, Thomas Scheper and Karl Schiigerl Institut
ftir Technische Chemie, Universitiit Hannover, Caliinstr. 3, D-3000 Hannover I (Germany)
Andreas Spreinat and Garo Antranikian Instiiur ftir Mikrobiologie,
Georg-August-Universittit
Giittingen, Grisebachstr. 8, D-3400 Giittingen (Germany)
Elisabeth Fraune B. Braun Melsungen AG, Schwarzenberger (Received
Weg 73, D-3508 Melsungen (Germany)
20th November
1990)
Abstract A system for real-time monitoring of specific proteins in fermentation processes is introduced. For measurements the turbidity caused by aggregates formed between the proteins to be detected and their antibodies is measured photometrically at 340 nm. The flow-injection analysis principle was used to automate the assay fully, including calibration and washing steps. The assay was intended for on-line product monitoring. It was therefore optimized to antibodies (mab) cover concentration ranges of l-1000 mg l- ‘. The analyser was used to measure monoclonal produced in fermentations of mouse-mouse hybridoma cells and to quantify pullulanase isoenzymes produced in a fermentation of Clostridium thermosuifurogenes. The fermentations took between 240 and 450 h, including extended phases of steady-state production. During that time, the long-term stability of the analyser system was excellent. A relative standard deviation of less than 2% was calculated for the data. A conventional ELISA served as a reference assay in the mab measurement. The enzymatic activity found in comparable off-line samples was used to correlate the on-line pullulanase measurements. Correlation coefficients between 0.94 and 0.99 were found between the on-line assay and the reference assays. Keywords:
Flow system;
Turbidimetry;
Bioprocess
monitoring;
The rapid development of biotechnological production methods over the last decade makes large-scale industrial production of biologically active substances conceivable. Close monitoring of the process is not only mandatory for substances to be used for therapeutic applications in humans, but also a necessary part of process optimization. Control and regulation of parameters such as tem-
’ Present address: Department of Chemical Engineering, University, New Haven, CT 06511, U.S.A.
0003-2670/91/$03.50
0 1991 - Elsevier
Process
analysis;
Proteins
perature, oxygen content and pH can be taken for granted in most modern fermentation processes. More and more analysers are being introduced for the monitoring of low-molecular-weight nutrients and metabolites [1,2]. Many of these analysers are fully automated and can be interfaced to a computer-based process control system. The substance produced, however, will often be a protein or other molecule of high molecular weight. Little has been done to permit the real-time determination of the concentration and stability of these products.
Yale
Science Publishers
Fermentation;
B.V.
114
The discriminatory power of antibodies has made immunoassay the most suitable candidate for this type of detection. Antibodies can be raised against virtually all proteins. The reaction between the antibody and the protein (antigen) is highly specific and sample preparation therefore becomes less complex. The assay described was designed to follow product formation rather than the build-up of some impurity. Concentrations of up to 1 g l- ’ had to be expected. In order to keep the system as simple as possible a turbidimetric immunoassay [3,4] with a detection range of 1 X 10P4-1 g 1-l was chosen, rather than an assay with a lower detection limit connected with a sophisticated appliance for sample dilution. The principle of flow-injection analysis (FIA) is the most important method for the automation of wet chemical analysis [5,6]. A given reaction does not have to reach equilibrium, as sample dispersion in an FIA system is highly reproducible. This is especially important for the automation of antibody-antigen reactions, which are based on the encounter of certain areas of large molecules [7]. The aim in this work was to establish a method for the on-line detection of specific proteins and to prove its potential for long-term monitoring during real fermentation processes. The analyser was successfully employed to monitor the production of monoclonal antibodies by hybridoma cells during two fermentations. A slightly modified assay was used to follow the production of a thermostable enzyme by bacteria.
EXPERIMENTAL
Flow-injection analyser for on-line detection of specific proteins A merging-zones stopped-flow FIA system as shown in Fig. 1 was assembled for assay automation. The different elements were activated by a set of interacting time-switch relays. The two loops for sample and sample blank were continuously flushed with fermenter supematant. Complete replacement of residual buffer was assumed after flushing the injector with ten loop volumes of sample. The reagent loop was filled discontinuously after displacement of residual buffer by an
R. FREITAG
ET AL.
1I
L-!-l
t
buffer
Fig. 1. Schematic diagram of the flow-injection analyser used to automate the turbidimetric immunoassay. The different elements of the system are controlled by a set of time-switch relays. The data are transferred to a computer for processing.
air bubble. Antibody consumption was thus reduced. Sample and reagent were injected into separate buffer streams. After merging, the flow was interrupted for a certain incubation time to allow the aggregates to form. After incubation the turbidity of the reagent mixture was measured. In the meantime a sample blank was established in the other section of the FIA manifold. Buffer vessels and reaction coils were thermostated at 37 o C. A detection wavelength of 340 nm (Scalar 6300 filter photometer) was chosen. This wavelength is far enough from the protein absorption maximum of 280 nm to ensure a low background noise, while the signal intensity is still high compared with readings taken at longer wavelengths. The data were transfered to a modified Atari computer and converted into antigen concentrations by means of a calibration graph [8]. Both peak height and area could be used for this purpose. The system was recalibrated at regular intervals. Washing steps, e.g., of the sampling line, could be included when necessary.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
115
PROCESSES
probe, the harvest pump drew the fermenter contents through a static filtration unit equipped with a hydrophilic microporous tubular membrane. As proteins and cells were not able to traverse the membrane, they were retained in the fermenter. Analysis. Off-line samples were taken daily and aliquots stored at -20” C for further analysis after clearing by centrifugation. Cell densities were established with a haemocytometer, using the Trypan Blue dye exclusion test to differentiate between viable and non-viable cells [lo]. The amino acid content of the fermenter supematant was measured by reversed-phase liquid chromatography after precolumn derivatization with o-phthaldialdehyde. Overall protein contents were determined according to Bradford [ll]. An automated analyser was connected to the CSTBR for glucose and lactate measurements (YSI 2000; Yellow Springs Instruments). A BIOPEM filtration module (B. Braum Diessel) was used to obtain a continuous sample stream [12]. A height-adjustable stirrer was placed above the filtration membrane to prevent filter-cake for-
High cell density perfusion culture of IgG-producing hybridoma cells Procedure. Mouse-mouse hybridoma cells (cell
line DB9G8, PTCC HB 124 [9]) were propagated in Dulbecco’s modified Eagle’s medium (DME) supplemented with controlled process serum replacement (CPSR) 4 (both from Sigma). The cells produce monoclonal antibodies (IgG,,, K-light chain) against human insulin. A 1.6-1 continuously stirred tank bioreactor (CSTBR) equipped with control mechanisms for temperature, pH and dissolved oxygen (Biostat MC; B. Braun Diessel) was used. The experimental set-up is shown in Fig. 2. The dissolved oxygen was controlled at 20% air saturation and the pH at 7.2. A temperature of 37O C was maintained throughout. Fresh culture medium was continuously pumped into the CSTBR during the perfusion phase of the experiment. Dilution rates were increased from l.Od-’ to 1.52d-’ during the first and from 0.8d-’ to l.ld-’ during the second fermentation according to the nutritional needs of the increasing cell population. Activated by a level
YSI
option
2000
II
!!
2
I lmmuno
1
FIA
feed
TFU
BIOPEM
lmmuno n FIA BIOPEM YSI
I > harvest
bioreactor
1 2000
8
option J
1
.
pump
Fig. 2. Perfusion bioreactor for the production of monoclonal antibodies by hybridoma cells. A Yellow Springs YSI 2000 analyser for glucose and lactate measurement and a flow-injection analyser for IgG measurements are connected to the fermenter via two sampling modules. For sampling the BIOPEM module is used either by itself or connected with a tangential filtration unit (TFU) as sterile barrier.
116
R. FREITAG
filtrate
outlet
inlet
Fig. 3. Tangential filtration with a 0.22~pm membrane the BIOPEM if necessary.
unit (TFU). The unit was equipped and used as a sterile barrier after
mation. If neccessary, a tangential filtration unit (TFU) (Fig. 3) was connected in series with the BIOPEM. The units were fitted with 0.22~pm PVDF (polyvinylidendifluoride) membranes (Durapore type; Millipore) or modified PTFE membranes of different pore sizes. Turbidimetric immunoassay (TIA) for measurements of monoclonal mouse antibody (mab). Anti-
mouse IgG (Sigma M-7023, developed in rabbit, titre 1.8 mg ml-‘) was used as a reagent. A 0.01 mol 1-i sodium phosphate buffer (pH 7.2) con-
ET AL.
taining 30 g 1-l PEG 6000 and 4.5 g 1-i sodium chloride (all from Fluka) was used throughout. The flow-rates of the buffer and sample streams were adjusted to 1 ml mm’. Reagent was pumped at 0.1 ml min- i. Injection loops of 0.05 ml were used in all instances. The detection range was l-1000 mg 1-i mab. A purified mouse IgG (Sigma I-5381) was used at several dilutions to obtain the calibration graph given in Fig. 4. An incubation period of 90 s was necessary, adding to the 150 s per analysis. During the experiments the system was recalibrated using standard IgG concentrations of 1,100 and 1000 mg 1-l. Frequent washing with Decon 90 (Zinser Analytik) was necessary to prevent protein build-up in the system. The analyser was automatically activated three times a day for a triple measurement. A conventional sandwich enzyme-linked immunosorbent assay (ELISA) was used as a reference assay for the TIA. Affinity-purified goat anti-mouse IgG was used both as a coating and as a detection anti-
.
360
720
Fig. 4. Calibration graph for the turbidimetric assay of monoclonal mouse antibodies. The signal intensities are given in ODs, units, since the term optical density can be used regardless of the physical phenomenon causing the observed decrease in light intensity. In our case this decrease is due to the scattering of the incoming light by the immunocomplexes, rather than to e.g. an absorbance of the light.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
body. For the latter purpose the antibody was conjugated to alkaline phosphatase. All reagents for the ELISA were obtained from Dianova (Hamburg), except the IgG standard (Sigma I5381) used for calibration. Continous culture of Clostridwm thermosulfurogenes Procedure. The production of three thermostable pullulanase isoenzymes by a Clostridium EMl, species (Clostridium thermosulfurogenes
Deutsche Sammlung ftir Microorganismen 3896) was monitored. A starch-limited medium [13] was utilized for cultivation. A 2-1 BCC fermenter (Schtitt, Gottingen) equipped with in-line sensors for temperature and pH and a deep tube for off-line sampling was used. The experiment was done under anaerobic conditions. The temperature was kept at 60” C throughout and the pH was regulated at 6.0 during the continuous culture by adding appropriate amounts of sterile 2 mol 1-l potassium hydroxide solution. A cross-flow hollow-fibre module (KF 200 010 S ME; Microgon) was connected to the
00
240
480 Pullulanase
720 [U/l]
117
PROCESSES
bioreactor to obtain a representative sample stream for the on-line analyser. Analysrs. Off-line samples were taken every 2 h during the batch phase and three times daily during the continuous culture. The samples were stored at 0” C for further analysis. Cell densities were determined by measuring the turbidity of the sample at 578 nm against cell-free fermenter supernatant. Pullulanase concentrations in the fermenter and in the on-line sample stream were measured according to Bergmeyer et al. [14]. Turbidlmetric Immunoassay (TIA) for pullulanase. The assay is to a large extent similar to
that described above for mouse IgG. Here 0.05 mol 1-l sodium phosphate buffer (pH 7.4) was used. The PEG content was increased to 40 g l- ’ while the sodium chloride concentration remained at 4.5 g 1-l. A longer incubation period of 120 s proved to be beneficial. Anti-pullulanase antibodies were obtained by inoculating a rabbit with purified pullulunase produced by C. thermosulfurogenes. The antibodies reacted in an Ouchterlony double diffusion test [15] with all pullulanase isoenzymes. The final antiserum was di-
960
1200
Fig. 5 Cabbratlon graph for the turbldlmetnc puIIulanase assay See also remark m legend to Rg 4
118
R. FREITAG
luted 1: 40 using the reaction buffer without PEG as dilutant. A detection range of 10-1000 U 1-l was established using purified pullulanase as a standard. The dose-response curve is given in Fig. 5. The system ‘was recalibrated using pullulanase concentrations of 0, 10, 100 and 1000 U 1-l.
RESULTS AND DISCUSSION
Fermentations of hybridoma cells Production with mammalian cells constitutes a as these major challenge in biotechnology, organisms require a highly controlled environment. Growth conditions and production rates of the hybridoma cell line HB 124 have been investigated in the Melsungen laboratory for some time [16]. The growth kinetics for the fermentations performed with on-line product monitoring did not differ significantly from those observed before. During the initial batch phase the cell density increased exponentially from 0.14 X lo6 to 1 x lo6 viable cells ml-‘. Exponential growth was maintained during the first 2 days of the perfusion
ET AL.
culture and the specific growth rate calculated was 1.2 d-l. Subsequently the culture entered a stationary phase. In both experiments final cell densities of ca. 2 x 10’ viable cells ml-’ were reached. During the steady phase, glucose and lactate levels could be stabilized at 2 and 1 g l-‘, respectively. Amino acid concentrations were held at constant levels of ca. 50% of the original medium concentrations. Glutamine and methionine concentrations were below 10%. As glutamine is important for cell metabolism [17], growth and production might be restricted under these conditions. Supplementing the regular medium with glutamine should therefore be beneficial. In a perfusion culture proteins are retained within the bioreactor [18,19]. The advantages of high cell and product concentrations are self-evident. Overall costs are lowered as the amount of serum or serum substitute used to enrich the culture medium can be gradually reduced during the fermentation. In this instance the CPSR 4 content was lowered from 4% at the beginning to 1.1% towards the end of the cultures. This also improves the protein to product ratio. The resulting
Time [h]
Fig. 6. Protein and monoclonal antibody concentrations during the original fermentation of HB 124 cells. The protein concentration of the fermenter supematant was measured according to Bradford [ll]; the antibody concentration was measured in centrifuged off-line samples using a sandwich ELISA for mouse IgG.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
mab and overall protein concentrations for the original fermentation can be taken from Fig. 6. IgG levels of 3% were reached at the end of the first and 4.68,at the end of the second fermentation. Although the fermentation process itself and the established analysis connected with it did not cause any difficulty, the acquisition of a representative sample was found to be a major obstacle m the on-line determination of proteins. For reasons of asepsis, the membrane dividing the nonsterile analyser and the culture vessel should not have pore sizes larger than 0.22 pm (sterile barrier). Several set-ups were studied during the original fermentation. The BIOPEM was fitted with a 0.22-, a 10 or a 5-pm membrane. If necessary, the TFA equipped with a 0.22~pm membrane was connected to it. Protein and mab concentrations were measured on both sides of all filtration membranes. Within 70-100 h the permeability of the BIOPEM membrane decreased to ca. 50%. The permeability for mab was usually better (ca. 80%). The decrease in permeability was found to be independent of the pore size; a 5-pm membrane did not perform better than a 0.22~pm membrane in the BIOPEM. Unfortunately, the stirrer in the BIOPEM did not prevent clogging and concentration polarization. A 0.22~pm membrane utilized as
PROCESSES
119
an additional sterile barrier after the primary sampling unit, however, demonstrated no further tendency to retain proteins. This emphazises the importance of cell debris and large membrane proteins for filter fouling. Although the large dead volume (200 ml) of the BIOPEM constituted another problem, the module was utilized again during another fermentation. The unit was fitted with a 0.22~pm membrane and exchanged daily. Adequate sampling thus became possible. The construction of a sampling device suitable for proteins, however, remains a challange to engineering. Product monitoring was archived during both experiments, despite the difficulties caused by the sampling procedure. The results are depicted in Fig. 7. During the original culture the mab concentrations did not increase as usual. This was to be expected, as the concentrations in the BIOPEM sample stream were not directely correlated with the mab level reached in the fermenter. Consequently, the behaviour became more normal during the second fermentation. The sharp decrease in mab concentration after 320 h is caused by dilution of the fermenter contents with fresh culture medium. A relative standard deviation of less than 2% was calculated for the measurements. If the product concentrations established with
140
200
300
400
500
Time [h] fig % C’oncentratlons he
DB9G8
ATCC
of monocional- anthodies HB 124 0 = Expenment I,
measured- turbldimetncally expenment II.
A =
diumg
two perfuslon
cultures
of hylindbma
cellq celI_
R FREITAG
120 250
++
+ ++ + +
+
50 4 *+ +* L.9 0
50
I. 100
I. 150
200
Mouse IgG [mgll], TIA Fig. 8. Correlation between mouse IgG measurements obtamed with the turbldlmetnc immunoassay and urlth the ELISA reference assay for samples with ldentlcal background The fermenter supernatant was clanfled by fdtratlon and samples were analysed m an off-hne mode with both methods A correlation coefflclent of 0 98 was calculated.
prolonged storage. with the former.
Process control
ET AL
is only possible
Fermentation of Clostrthum thermosulfurogenes Protein production by rmcroorganisms such as bacteria is more important than production by mammalian cells, as these organisms are less demanding and easier to propagate. The production of a Clostndzum species was followed by a specific immunoassay. The bacteria excrete three isoenzymes of a thermostable pullulanase [20]. Pullulanases are debranching enzymes capable of hydrolysing a-1,6-glycosidic linkages in lngh-molecular-weight polymers of D-glucose. The thermostability of the pullulanases produced by C. thermosulfurogenes in connection with the pullulytic ability makes these enzymes extremely interesting to the food industry.
0
CI
Pullulanase [U/I], Fermentationbroth centrituged 80 160 320 240 I
I
I
400
I
4
4
9
the TIA and the ELBA are compared, good agreement is found for concentrations below 15 coefficient of 0.94 was mg 1-l. A correlation calculated. For higher concentrations the correlation became worse. During the onginal experiment a correlation coefficient of 0.83 was found. The correlation improved for the second fermentation, a value of 0.96 being obtained. Generally, concentrations ascertained with the ELBA are lower than those produced by the TIA. A certain amount of protein deterioratron must be assumed during sample storage prior to the ELISA measurements. When the TIA was employed m an off-line mode to analyse samples after storage, the correlatton with the ELISA measurement was better, as shown in Fig. 8 (correlation coefficient 0.98). Although the problem of obtaining a representative sample remains unsolved, the amount of time and the manual labour involved in product monitoring were considerably reduced. Mab concentrations measured in fresh fermenter supernatants should be superior to those obtained after
4 <\
E
16I -
z t I= 27
36
45 0
80
160
240
Puklanase
[U/l], TIA
320
400
Fig 9. Values for pullulanase concentrations measured turbtdlmetncally and with the enzymatic actlvlty reference assay dunng the first 20 h of C thermosulfurogenes fermentation Fermenter supematant samples were centnfuged and ahquots used for both types of measurements.
REAL-TIME
MONITORING
OF SPECIFIC
PROTEINS
IN FERMENTATION
Pullulanase monitoring proved to be much simpler than IgG monitoring. During the batch culture the automatic analyser was not activated. Instead, aliqupts of samples taken off-line were cleared by centrifugation and injected manually into the TIA system. Correlation with the activity measurements was good, as can be deduced from Fig. 9. A correlation coefficient of 0.99 was calculated. In the continuous phase a single cross-flow hollow-fiber filtration module was employed for the entire 244-h fermentation. During that time no significant difference was observed between the pullulytic activity of samples taken before and after the module. The continous culture was started after 20 h, when the pH of the fermenter content had decreased to 4.01. A dilution rate of 0.075 h-’ was adjusted initially. After 80 h (4.5 residence times), an equilibrium between bacteria growth and production rates and the dilution rate was reached. After four more residence times the dilution rate was raised to 0.1 h-i. The cell density did not stabilize again, but a new plateau for pullulanase was archived. Pullulanase production is induced by a high-
.
800
W _ F 2,
600
PROCESSES
121
molecular-weight polymer of D-glucose as main source of carbohydrate in the culture medium. If this source is replaced with glucose itself, pullulanase production ceases. Such an exchange from a starch to a glucose basis took place after 200 h. After 244 h the pullulanase concentration had decreased to zero and the experiment was terminated. Cell growth and pullulanase production as measured by the TIA and the activity assay are shown in Fig. 10. A correlation coefficient of 0.94 was calculated for the two assays. The relative standard deviation for the TIA was again below 2%. The detection limit of 10 U 1-l constituted no problem as this concentration was surpassed after 10 h of fermentation. The longterm stability of the system was excellent. Repeated recalibrations showed no major shift in the calibration graph. During the initial batch culture, the higher sensitivity of the enzyme activity assay makes that method preferable, especially as process control is not possible at this point. Once a stable steady state has been reached the continuous production of pullulanase can continue for several months. In
z
W 2 f
ti
400
i z 200
0
0
50
150
100
200
TIME [h] Fig. 10. Cell growth activity assay.
and pullulanase
concentration
measured
on-line
with the turbidimetric
assay
and off-line
with the enzyme
122
R. FREITAG
this phase, when the detection limit is no problem, the immunoassay will simplify process maintenance and safety supervision.
Conclusions The industrial-scale production of biologically active compounds demands the development of new techniques for their on-line assessment. Experimental results indicate that the FIA immunoassay system introduced here allows on-line monitoring of product formation in continuous long-term cultivation processes. The inclusion of high-molecular-weight substances in the group of process control parameters thus becomes feasible. This work was supported by a DECHEMA grant to R. Freitag, grant No. 0318815A from the Bundesministerium ftir Forschung and Technologie, and by the BMFT within the Schwerpunkt: Grundlagen der Bioprozesstechnik.
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ET AL.
3 G.A. Hudson, R.F. Ritchie and J.E. Haddow, Clin. Chem., 27 (1981) 1838. 4 P.J. Worsfold and A. Hughes, Analyst, 109 (1984) 339. 5 J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 114 (1980) 19. 6 W.E. van der Linden, Anal. Chim. Acta, 179 (1986) 91. 7 W.R. Griswold, J. Immunoassay, 1 (1987) 145. 8 R. Freitag, Th. Scheper and K. Schligerl, Anal. Chem., submitted for publication. 9 J. Schroer, T. Bender, R.J. Feldmann and K.J. Kim, Eur. J. Immunol., 13 (1983) 693. 10 W. Halle, Zell- und Gewebeziichtung bei Tieren, Gustav Fischer, Stuttgart, 1976. 11 M.M. Bradford, Anal. B&hem., 72 (1976) 248. 12 K.-H. Kroner, W. Stach and W. Kuhlmann, Chemie-Technik, 15 (9) (1986) 74. 13 G. Antranikian, P. Zablowski and G. Gottschalk, Appl. Microbial. Biotechnol., 27 (1987) 75. 14 H.U. Bergmeyer, K. Gawehn and M. Grabe, in H.U. Bergmeyer (Ed.), Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 3. Auflage, 1974. 15 6. Ouchterlony, Acta Pathol. Microbial. Stand., 26 (1949) 507. 16 E. Fraune, Ch. Fenae and W. Kuhlmann. 8th International Biotechnology Symposium, Paris, 1988, Abstract 115. 17 L.J. Reitzer, B.M. Wice and D. Kenell, J. Biol. Chem., 254 (1979) 2669. 18 Y. Takazawa, M. Tokashiki, K. Hamamoto and H. Murakami, Cytotechnology, 1 (1988) 171. 5 (1989) 48. 19 D. Siewert, BioEngineering, C. Herzberg and G. Gottschalk, Appl. 20 G. Antranikian, Environ. Microbial., 53 (1987) 1668.