- Email: [email protected]
Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies
Process Biochemistry 34 (1999) 159 – 165
Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies J. Feuser a, M. Halfar a, D. Lu¨tkemeyer b, N. Ameskamp b, M.-R. Kula a, J. Tho¨mmes a,* a
Institut fu¨r Enzymtechnologie, Heinrich-Heine Uni6ersita¨t Dusseldorf, 52426 Ju¨lich, Germany b Uni6ersita¨t Bielefeld, Technische Fakulta¨t, 33501 Bielefeld, Germany Received 6 May 1998; accepted 31 May 1998
Abstract The interaction of a mammalian cell culture broth with two commercially available adsorbents for the use in expanded bed adsorption (EBA) has been studied. A cation exchange resin (Streamline SP) and an affinity adsorbent (Streamline rProtein A) were compared with regard to adsorption of hybridoma cells during sample application as well as potential cell damage. The results showed that hybridoma cells interact significantly with an expanded bed of cation exchange adsorbents but not with the Protein A adsorbent. After application of 17–20 sedimented bed volumes a saturation of the Streamline SP resin with cells was noted. With both adsorbents no measurable cell damage was found and IgG1 was recovered in approximately 95% yield. The capacity for IgG1 adsorption at 3% breakthrough was 2.7 mg IgG1/ml Streamline rProtein A at a constant fluid velocity of 380 cm/h and 1.0 mg IgGl/ml Streamline SP at 215–240 cm/h fluid velocity. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Expanded bed adsorption; Monoclonal antibody; Protein purification; Ion exchange chromatography; Affinity chromatography; Hybridoma cells
Nomenclature F 0 Bo U L Daxl t t
normalized effluent concentration ( — ) normalized effluent time ( — ) Bodenstein number ( — ) superficial fluid velocity (m/s) bed length (m) coefficient of axial mixing in the liquid phase (m2/s) mean residence time (s) time (s)
1. Introduction Expanded bed adsorption (EBA) has been introduced as a primary recovery operation to handle unclarified * To whom all correspondence should be addressed. E-mail: [email protected].
culture broth. By fluidizing suitable adsorbent particles, the increased void fraction within the column allows the application of particle containing feedstock and thus yields a combination of clarification, concentration and adsorptive purification in a single step [1–3]. Various applications of this technology have been published, including secreted proteins from Escherichia coli [4] and yeast cells [5,6], proteins from E. coli lysate [7] and yeast homogenates [8], monoclonal antibodies [9,10], and proteins from milk [11]. The current literature on EBA has been mainly restricted to the description of successful applications as well as to studies investigating the influence of operating parameters on the adsorption of model proteins from aqueous solutions [12–14]. It was found that similar to protein adsorption to packed beds of porous media, the performance of EBA is controlled by transport processes. As an additional limitation, the mobility of the fluidized adsorbents has to be taken into account in terms of increased axial mixing within the bed. Axial mixing in
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 8 3 - 1
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J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
expanded beds is not critical during adsorption of model proteins from aqueous solutions as long as a minimum settled bed height and a minimum fluid velocity are maintained [14]. Systematic investigations on the influence of crude feedstock on the sorption performance are still scarce. These data, however, are very important in order to assess the suitability of EBA for the industrial purification of proteins. Reliable applications require a stable process, a demand that can be severely compromised by interactions of biomass in real feedstock with the expanded bed. Potential interactions include the adsorption of cells or cell components to the stationary phase, which can reduce the sorption efficiency by hindering the transport of adsorbate to and within the porous resin. More severe fouling induces aggregation of adsorbent particles, which then can lead to the formation of dead water zones and flow channels within the expanded bed. Flow maldistribution will lead to a reduced availability of the adsorbent particles in the bed and have consequences on the static capacity. Furthermore, axial mixing will be increased with the respective consequences for the kinetics of the sorption process. In the worst case, the expanded bed may break down completely because of aggregation effects. Adsorbed biomass may co-elute with the target protein during desorption, which will complicate further downstream processing steps. A second important aspect is the potential damage of cells during their passage through the expanded bed. In the case of secreted proteins, cell damage will release intracellular components such as nucleic acids or proteins, which in turn contaminate the product. In this paper, an attempt is made to assess the problems outlined above by using monoclonal antibody purification (MAb) with EBA based on two different adsorption mechanisms. Monoclonal antibodies are a relevant product in the pharmaceutical industry and are produced on a large scale. As they are used for diagnostic and therapeutic purposes, contamination by nucleic acids is an important issue. Hybridoma cells lack a stable cell wall and are generally sensitive to shear [15]. They are thus an adequate test system for potential cell damage in the expanded bed. The two most commonly used adsorption procedures for MAbs are cation exchange and protein A affinity adsorption. In this study Streamline SP and Streamline rProtein A were used in an EBA process in order to investigate the interaction of hybridoma cells with the two resins as well as the influence of passage of the cells through the bed on viability. Two intracellular markers (lactate dehydrogenase and total DNA content) were chosen as a measure for cell damage. Thc performance of the adsorption process was charactcrized by the breakthrough of cells and IgG1 respectively, during sample application.
2. Materials and methods
2.1. Cells The cell line used in these experiments was a mouse/ mouse-hybridoma for production of monoclonal IgG1.
2.2. Culti6ation A bioreactor of 100-litre total volume (Biostat UD100, B. Braun Biotech International) with bubblefree aeration was used for the pilot scale cultivation of hybridoma cells. The growth medium contained the following proteins: human serum albumin (500 mg/l); human transferrin (10 mg/l); and bovine insulin (10 mg/1). A detailed description of the ingredients and the cultivation procedure is given elsewhere [16]. A single batch was used in the experiments.
2.3. Expanded bed adsorption process A Streamline 50 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for the studies (50 mm diameter; 1000 mm length). The experimental setup consisted of two Watson-Marlow Type X100 peristaltic pumps (Watson-Marlow Ltd, Falmouth, UK) with a 4.5-mm internal diameter Marprene synthetic rubber tubing. The UV absorbance for protein detection of the stream leaving the column was measured in a flow spectrometer (Knauer, Berlin, Germany) at 280 nm and was monitored by a X-Y-recorder (Kipp und Zonen, Solingen, Germany).
2.4. Cation exchange Streamline SP (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was filled into the column to a sedimented bed height of 33 cm (650 ml adsorbent volume). Equilibration was carried out for 50 min at 240 cm/h to result in a 2.5-fold bed expansion. The following buffers were employed: 1. Dilution buffer: 10 mM potassium phosphate-buffer (pH 5.0) 2. Equilibration buffer: 50 mM potassium phosphatebuffer (pH 5.5) 3. Wash buffer: 50 mM potassium phosphate-buffer (pH 5.5) 4. Elution buffer: 20 mM potassium phosphatebuffer+ 1 M NaCl (pH 8.0) The 20-litre harvest was diluted to 40 litre with dilution buffer and adjusted to pH 5.5 with 10% phosphoric acid. This was performed slowly with stirring to prevent local concentration peaks resulting in undesired cell damage. The cell concentration after dilution was 1.2×l06 cells per ml at 89% viability. The suspension was stirred at 80 rpm, applying a total volume of 32
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
litre to the column. During sample application, the flow was reduced from 240 cm/h to 180 cm/h in order to maintain the 2.5-fold bed expansion.
2.5. Affinity adsorbent Streamline rProtein A (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used at a bed height of 22.5 cm (450 ml adsorbent volume) in a Streamline 50 column. The fluid velocity was kept constant at 380 cm/h resulting in a bed expansion of 2.7-fold at the beginning and 2.9-fold at the end of the feeding phase. The following buffers were employed: 1. Equilibration: 50 mM potassium phosphate-buffer (pH 7.5) 2. Wash: 50 mM potassium phosphate-buffer (pH 7.0) Elution 100 mM sodium citrate-buffer (pH 4.0) The feedstock was slowly adjusted to pH 7.5 with a 10% sodium hydroxide solution and stirred in a tank at 60 rpm. The total feed volume amounted to 66 litres.
2.6. Analytical methods Off-line samples of the effluent and the feedstock were taken at intervals of 45 min during the feeding phase. The concentration of cells, intra- and extracellular LDH and extracellular DNA were analysed in all samples.
2.7. Cell concentration offline Cell concentration and viability in culture broth samples were determined by trypan blue dye exclusion (Fluke, Neu-Ulm, Germany) in a Neubauer haemocytometer.
2.8. Wash out of cells In order to determine hybridoma cell desorption from the saturated resin, the optical density of the effluent in the wash step was monitored online using a flow spectrometer (Knauer, Berlin, Germany) at 600 nm. The linear range of detection was up to a cell concentration of 2× 106 cells/ml.
2.9. Lactate dehydrogenase acti6ity The extent of cell lysis was determined by measuring lactate dehydrogenase (LDH) activity by reduction of pyruvate to lactate, which is coupled to the oxidation of NADH to NAD [17]. The reaction mixture contained PBS, pH 7.4, pyruvate, NADH, and depending on the sample 100–300 ml of test solution in a volume of l ml. Absorbance was monitored at 340 nm for 2 min at a scanning rate of 50 scans/min using a spectrophotometer (UVIKON 930, Kontron, Neufahrn, Germany).
161
LDH activity was calculated from the linear regression curve using the molar extinction coefficient of NADH of 6300 M − 1 cm − 1. Maximum LDH activity in the samples was obtained after disruption of cells by sonification.
2.10. DNA concentration DNA concentration was measured with an intercalating fluorescent dye (Pico Green P-7581, Molecular Probes Inc., USA). An aliquot of 100 ml of Pico Green was diluted in 20 ml, 10 mM Tris–HCL with 1 mM EDTA pH 7.5. From this solution, 500 ml was mixed with 500 ml of sample. After an incubation of 3 min in the dark the samples were measured in a fluorescence spectrofluorometer exited at 480 nm and the fluorescence intensity was measured at 520 nm. A calibration curve was obtained by measuring calf thymus DNA.
2.11. Product concentration MAb concentration was detected with a standard sandwich-ELISA assay as described by Ray et al. [18].
3. Results and discussion
3.1. Cell retention One of the main interaction problems between biomass present in the broth and the adsorbent is the retention of cells and/or the formation of cell-adsorbent aggregates during sample application. Analogous to protein adsorption, cell retention during sample application in an EBA process can be described by a breakthrough curve. The cell concentration in the effluent can be measured and plotted as c/co versus the normalized effluent time (U = t/t). If no interaction is present, cell breakthrough should coincide with the breakthrough of a tracer (e.g. acetone). This hydrodynamic breakthrough can be simulated by the dispersion model (eqn (1)) with the dimensionless Bodenstein number, Bo, describing axial mixing [19]. TypTable 1 Concentration and amount of DNA in the feedstock, effluent and eluate of Streamline SP and Streamline rProtein A EBA DNA
Feed Effluent Elution
Streamline r Protein A
Streamline SP
Mean DNA conc. (ng/ml)
Total DNA (mg)
Mean DNA conc. (ng/ml)
Total DNA (mg)
3748 3648 903
254.9 248.1 0.6
1032 744 14016
33.0 23.8 10.3
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
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Fig. 1. Normalized hybridoma cell concentration in the effluent of the cation exchange expanded bed adsorption during sample application. Determination of the experimental data (open triangle) by haemocytometer counting. The line represents a simulation of breakthrough under non-binding conditions using eqns (1) and (2), Bo = 60.
Fig. 2. Normalized hybridoma cell concentration in the effluent of rProtein A expanded bed adsorption during sample application. Determination of the experimental (open triangle) data by haemocytometer counting. The line represents a simulation of breakthrough under non-binding conditions using eqns (1) and (2), Bo = 60.
ical values for Bo in EBA systems of the configuration used here are in the order of magnitude of 60 – 80.
homogenization of the broth is necessary in order to release the product. A disruption of cells before or during thc adsorption step is undesirable because this would additionally contaminate the supernatant and hamper the efficiency of the subsequent purification steps. Furthermore, released substances may serve as mediators of cell adsorption to the stationary phase. Thus cell/adsorbent interactions always have to be examined in combination with potential cell damage. In these experiments, three parallel measures of culture viability were employed: trypan blue dye exclusion, LDH and DNA [20] release by the cells. These assay techniques were used to determine the amount of damage to the cells that has occurred during storage in the feedstock as well as by passing the pump and the expanded bed system. The release and uptake of the tracers is affected by membrane permeability and expresses the viability of cells. The three techniques were used in parallel because of the possibility that the experimental conditions may bias the assays. In contrast to LDH and DNA measurements, the haemocytometer technique does not allow the quantification of shredded cells as they can occur at high shear forces or by autolysis. The difference of molecular weight of the tracers (trypan blue Mr = 960.8, LDH Mr = 140 000) may also lead to a variance of results depending on the extent of membrane damage. Furthermore, the released DNA may interact with the adsorbents indicating an influence on fluidization stability. Differentiation between dead and total cell concentration is feasible by quantifying LDH activity in the supernatant as well as in the cell lysate. Fig. 3 presents the LDH activity during sample application to Streamline SP. A comparison of the supernatant data indicates
< < ' ==
c (1 − U) F = =0.5 1−erf c0 U 2 Bo U*L Bo= Daxl
(1)
(2)
The cell concentration in the effluent of the cation exchange adsorption is compared with the hydrodynamic breakthrough curve in Fig. 1. The area between these two curves represents the quantity of the retained hybridoma cells during the application within 7 – 8 residence times. Beyond this point almost the same concentration of cells can be found in the effluent as in the feed, so no additional retention takes place and the matrix apparently is saturated with biomass. Different results were obtained when hybridoma culture broth was applied to the affinity adsorbent as shown in Fig. 2. No cell retention was evident during sample application over the first 15 residence times. The decreasing cell concentration appearing in the effluent during the late feeding phase, was not caused by the adsorbent matrix. The long feeding time of 10 h for a 66-litre culture broth caused accumulation of cells at the lower distributor net (stainless steel, 70 mm mesh size) and cells were thus mechanically filtered out from the feed solution, as found by visual inspection after the experiment was completed.
3.2. Cell damage Hybridoma cells secrete IgG1 into the medium, no
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
Fig. 3. Activity of lactate dehydrogenase (LDH) in the feed and effluent of the cation exchange expanded bed adsorption process; (closed square) activity of the feedstock after cell disruption; (open square) activity of the effluent after cell disruption; (closed triangle) extracellular activity of the feedstock; (open triangle) extracellular activity of the effluent.
that no detectable damage to the cells during passage through the expanded bed system has occurred. Cell lysis, however, can be observed in the feedstock tank. Large shifts in pH and dilution prior to sample application have important effects on culture sensitivity to shear and autolysis and may be seen as the reason for a steadily decreasing viability of cells in the feedstock [15]. The total volumetric LDH activity after cell disruption was proportional to the total cell concentration. Thus the difference between LDH activity after cell disruption in the feed and the value in the effluent from the EBAcolumn is an additional measure of cell retention. The LDH data coincide satisfactorily with the haemocytometer results in Fig. 1. This implies that the determination of LDH activity in the supernatant and in the cell lysate is sufficient for both the analysis of cell retention, and detection of cell damage. It is important, however, that this protein is not adsorbed by the resin under the conditions of sample application. LDH measurements in the supernatant during adsorption to the rProtein A matrix revealed no detectable damage of the hybridoma cells during a single passage through the bed (Fig. 4). Major cell lysis was again observed in the feedstock tank. LDH activity increased after 20 column volumes had been applied. In this case, the long residence time of the culture broth in the feed tank without a supply of oxygen also negatively influenced cell viability.
3.3. DNA contamination The concentration of nucleic acids is of major concern
163
Fig. 4. Extracellular activity of LDH in Streamline rProtein A EBA; (triangle) feedstock; (square) effluent.
in pharmaceutical products, as the limit of DNA exposure to patients is l00 pg/dose per day [21]. If DNA is not bound to the adsorbent, its concentration can also be used to detect the rate of cell damage as described above. Thc graphs in Fig. 5 reveal DNA adsorption onto Streamline SP, especially during the first 30% of sample application. An analysis of the eluate shows a nearly 3-fold reduction in the total DNA load. About 15 mg DNA/ml adsorbent was bound and co-eluted during IgG1 desorption. Streamline rProtein A showed no detectable affinity for DNA and the overall DNA content of the sample containing monoclonal antibody could be reduced 290-fold during the EBA process (Table 1).
Fig. 5. Concentration of DNA in the supernatant during adsorption of MAb on Streamline rProtein A (triangle) and Streamline SP (square), open symbols refer to feedstock, closed symbols refer to effluent.
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
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Table 2 Concentration and amount of IgG1 in the feedstock, effluent and eluate of Streamline SP and Streamline rProtein A EBA MAB
Feed Effluent Elution
Streamline r Protein A
Streamline SP
Mean IgG1 conc. (mg/ml)
Total IgG1 (mg)
Mean IgG1 conc. (mg/ml)
Total IgG1 (mg)
50.0 14.0 3667.5
3400 954 2347
21.0 0.84 826.2
672.0 26.9 607.2
3.4. Product reco6ery A comparison of capture step efficiency of both matrices is summarized in Table 2. The breakthrough capacity of IgGl at 10% breakthrough (protein effluent concentration equal to 10% of the feed concentration) was 3.3 mg IgG1/ml Streamline rProtein A. In the experiment using Streamline SP the breakthrough of IgG1 did not exceed 2.5%. In both experiments, the yield of IgG1 was 95% and no IgG1 was found in the wash fraction. The elution volume for the affinity adsorbent was 1.42 bed volumes (sedimented), and 1.13 bed volumes for the cation exchange application.
3.5. Cell wash out The delayed cell breakthrough during EBA is either caused by mechanical retardation or by adsorption of biomass onto the surface of the adsorbent. In order to differentiate between these two phenomena, the effluent
Fig. 6. Online measurement of hybridoma cell concentration in the effluent during washing procedure of Streamline SP EBA; (thin line) 65% viability ; (intermediate line thickness) 0% viability ; (thick line) simulation under non-binding conditions using eqns (1) and (2), Bo= 60.
of the wash step was monitored with a flowthrough spectrophotometer. Optical density measurements at a wavelength of 600 nm are proportional to cell concentration of up to 2× 106 cells/ml and can give online information on cell clearance from the matrix during the washing procedure. In Fig. 6 the wash out behaviour from fluidized Streamline SP saturated with biomass of different viability is compared with the wash out function of a tracer under similar conditions as during sample application (Bol=60). The coincidence of the curves clearly demonstrates that the cells are adsorbed to the matrix and cannot simply be washed from the void volume of the bed.
4. Conclusions Expanded bed adsorption proved to be a gentle technique for the recovery of proteins from whole culture broth. This is of special interest for secreted products, as in the case of IgG1, when an additional cell lysis should be avoided in order to minimize contamination. The examined hybridoma cells did not exhibit any detectable cell damage. Taking into account that mammalian cells are one of the most shear sensitive organisms used in biotechnological production, it is a realistic assumption that neither bacteria nor yeast cells will experience damage caused by the passage of cells through the expanded bed or in the pumps used. Cell lysis resulting from unfavourable conditions in the feedstock tank can be reduced by minimizing the residence time of culture broth adjusted to feeding conditions. This could be achieved by inline dilution and pH adjustment in a relative small mixing tank situated between the cultivation reactor and the EBA system. A prerequisite for large scale applications is the reliability of the process. Biomass and DNA adsorption onto the resin beads is one of the major reasons for fluidization instability. Therefore it is of importance to quantify these parameters when investigating a new application for EBA. While adsorption of hybridoma cells was not detected with Streamline rProtein A and DNA adsorption was negligible, Streamline SP presented a distinct adsorption of cells onto the matrix that could not be desorbed in a conventional wash step. In addition, 16 mg DNA/ml Streamline SP was bound and co-eluted with the protein product. Cell adsorption to Streamline SP resins was also observed with other hybridoma cultures. The reason for this as well as the cause for DNA adsorption remains to be investigated. These aspects in combination with a satisfying breakthrough capacity of the affinity adsorbent suggest the use of Streamline rProtein A in an EBA mode as a
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
reliable initial antibodies.
purification
step
for
monoclonal
References [1] Chase. H. A., Purification of proteins by adsorption chromatography in expanded beds. Trends in Biotechnology 1994. 12, 296 – 303. [2] Hjorth, R., Expanded bed adsorption in industrial bioprocessing: recent developments. Trends in Biotechnology 1997, 15(6), 230 – 235. [3] Tho¨mmes, J., Fluidized bed adsorption as a primary recovery step in protein purification. Ad6ances in Biochemical Engineering/Biotechnology 1997, 58, 185–230. [4] Hansson, M., Stahl, S., Hjorth. R., Uhlen, M. and Moks, T., Single-step recovery of a secreted recombinant protein by expanded bed adsorption. Bio/Technology 1994, 12, 285–288. [5] Zurek, H., Kubis, E., Keup, P., Hoerlein, D., Beunink, H., Tho¨mmes, J., Kula, M. R., Hollenberg, C. P. and Gellissen, G.. Production of two aprotinin variants in Hansenula polymorpha. Process Biochemistry 1996, 31, 679–689. [6] Noda, M., Sumi, A., Ohmura. T. and Yokoyama, K, Process for purifying recombinant human serum albumin. European patent EP 0 699 687, 1996. [7] Barnfield-Frej, A.-K., Hjorth, R. and Hammarstroem, A., Pilot scale recovery of recombinant annexin V from unclarified E. coli homogenate using expanded bed adsorption. Biotechnology and Bioengineering 1994, 44, 922–929. [8] Chang, Y. K and Chase, H. A., Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption. Biotechnology and Bioengineering 1996, 49, 204 – 216. [9] Tho¨mmes, J., Halfar, M., Lenz, S. and Kula, M.-R., Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption. Biotechnology and Bioengineering 1995, 45, 203–211. [10] Batt, B. C., Yabannavar, V. M. and Singh, V., Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth. Bioseparation 1995, 5, 41–52.
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[11] Noppe, W., Hanssens, I. and De Cuyper, M., Simple two step procedure for the preparation of highly active pure equine milk lysozyme. Journal of Chromatography 1996, 719, 327 – 331. [12] Hjorth, R., Kaempe, S. and Carlsson, M., Analysis of some operating parameters of novel adsorbents for recovery of proteins in expanded beds. Bioseparation 1995, 5, 217– 223. [13] Chang, Y. K. and Chase, H. A., Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance. Biotechnology and Bioengineering 1996, 49, 512– 526. [14] Karau, A., Benken, C., Tho¨mmes, J. and Kula, M.-R., The influence of particle size distribution and operating conditions an the adsorption performance in fluidized beds. Biotechnology and Bioengineering 1997, 55, 54 – 64. [15] Pertersen, J. F., McIntire, L. V. and Papoutsakis, E. T., Shear sensitivity of cultured hybridoma cells (CRL-8018) depends on mode of growth, culture age and metabolite concentration. Journal of Biotechnology 1988, 7, 229 – 246. [16] Ameskamp, N., Lu¨tkemeyer, D., Tebbe, H. and Lehmann, J., Downstream processing im Pilotmabstab: Direkte affinita¨tschromatographische Aufreinigung monoklonaler Antiko¨rper aus Hybridomakulturen im stabilen Fliebbett. Bioscope 1997, 6, 14 – 21. [17] Legrand, C., Bour, J. M., Jacob, C., Capiaumont, J. and Martial, A., Lactate dehydrogenase (LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells as marker. Journal of Biotechnology 1992, 25, 231 – 243. [18] Ray, N. G., Karkare, S. B. and Runstadler, P. W., Cultivation of hybridoma cells in continuous culture: kinetics of growth and product formation. Biotechnology and Bioengineering 1989, 33, 724 – 730. [19] Fitzer, E., Fritz, W. and Emig, G., Technische Chemie. Springer Verlag, Heidelberg, 1995, pp. 301 – 303. [20] Wittler, J., Entwicklung einer fluorimetrischen Methode zur quantitativen Bestimmung von DNA in Zellkulturu¨bersta¨nden. Diplomarbeit, University of Bielefeld, Germany, 1996. [21] Berthold, W. and Walter, J., Protein purification: aspects of processes for pharmaceutical products. Biologicals 1994, 22, 135 – 150.
Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies J. Feuser a, M. Halfar a, D. Lu¨tkemeyer b, N. Ameskamp b, M.-R. Kula a, J. Tho¨mmes a,* a
Institut fu¨r Enzymtechnologie, Heinrich-Heine Uni6ersita¨t Dusseldorf, 52426 Ju¨lich, Germany b Uni6ersita¨t Bielefeld, Technische Fakulta¨t, 33501 Bielefeld, Germany Received 6 May 1998; accepted 31 May 1998
Abstract The interaction of a mammalian cell culture broth with two commercially available adsorbents for the use in expanded bed adsorption (EBA) has been studied. A cation exchange resin (Streamline SP) and an affinity adsorbent (Streamline rProtein A) were compared with regard to adsorption of hybridoma cells during sample application as well as potential cell damage. The results showed that hybridoma cells interact significantly with an expanded bed of cation exchange adsorbents but not with the Protein A adsorbent. After application of 17–20 sedimented bed volumes a saturation of the Streamline SP resin with cells was noted. With both adsorbents no measurable cell damage was found and IgG1 was recovered in approximately 95% yield. The capacity for IgG1 adsorption at 3% breakthrough was 2.7 mg IgG1/ml Streamline rProtein A at a constant fluid velocity of 380 cm/h and 1.0 mg IgGl/ml Streamline SP at 215–240 cm/h fluid velocity. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Expanded bed adsorption; Monoclonal antibody; Protein purification; Ion exchange chromatography; Affinity chromatography; Hybridoma cells
Nomenclature F 0 Bo U L Daxl t t
normalized effluent concentration ( — ) normalized effluent time ( — ) Bodenstein number ( — ) superficial fluid velocity (m/s) bed length (m) coefficient of axial mixing in the liquid phase (m2/s) mean residence time (s) time (s)
1. Introduction Expanded bed adsorption (EBA) has been introduced as a primary recovery operation to handle unclarified * To whom all correspondence should be addressed. E-mail: [email protected].
culture broth. By fluidizing suitable adsorbent particles, the increased void fraction within the column allows the application of particle containing feedstock and thus yields a combination of clarification, concentration and adsorptive purification in a single step [1–3]. Various applications of this technology have been published, including secreted proteins from Escherichia coli [4] and yeast cells [5,6], proteins from E. coli lysate [7] and yeast homogenates [8], monoclonal antibodies [9,10], and proteins from milk [11]. The current literature on EBA has been mainly restricted to the description of successful applications as well as to studies investigating the influence of operating parameters on the adsorption of model proteins from aqueous solutions [12–14]. It was found that similar to protein adsorption to packed beds of porous media, the performance of EBA is controlled by transport processes. As an additional limitation, the mobility of the fluidized adsorbents has to be taken into account in terms of increased axial mixing within the bed. Axial mixing in
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 8 3 - 1
160
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
expanded beds is not critical during adsorption of model proteins from aqueous solutions as long as a minimum settled bed height and a minimum fluid velocity are maintained [14]. Systematic investigations on the influence of crude feedstock on the sorption performance are still scarce. These data, however, are very important in order to assess the suitability of EBA for the industrial purification of proteins. Reliable applications require a stable process, a demand that can be severely compromised by interactions of biomass in real feedstock with the expanded bed. Potential interactions include the adsorption of cells or cell components to the stationary phase, which can reduce the sorption efficiency by hindering the transport of adsorbate to and within the porous resin. More severe fouling induces aggregation of adsorbent particles, which then can lead to the formation of dead water zones and flow channels within the expanded bed. Flow maldistribution will lead to a reduced availability of the adsorbent particles in the bed and have consequences on the static capacity. Furthermore, axial mixing will be increased with the respective consequences for the kinetics of the sorption process. In the worst case, the expanded bed may break down completely because of aggregation effects. Adsorbed biomass may co-elute with the target protein during desorption, which will complicate further downstream processing steps. A second important aspect is the potential damage of cells during their passage through the expanded bed. In the case of secreted proteins, cell damage will release intracellular components such as nucleic acids or proteins, which in turn contaminate the product. In this paper, an attempt is made to assess the problems outlined above by using monoclonal antibody purification (MAb) with EBA based on two different adsorption mechanisms. Monoclonal antibodies are a relevant product in the pharmaceutical industry and are produced on a large scale. As they are used for diagnostic and therapeutic purposes, contamination by nucleic acids is an important issue. Hybridoma cells lack a stable cell wall and are generally sensitive to shear [15]. They are thus an adequate test system for potential cell damage in the expanded bed. The two most commonly used adsorption procedures for MAbs are cation exchange and protein A affinity adsorption. In this study Streamline SP and Streamline rProtein A were used in an EBA process in order to investigate the interaction of hybridoma cells with the two resins as well as the influence of passage of the cells through the bed on viability. Two intracellular markers (lactate dehydrogenase and total DNA content) were chosen as a measure for cell damage. Thc performance of the adsorption process was charactcrized by the breakthrough of cells and IgG1 respectively, during sample application.
2. Materials and methods
2.1. Cells The cell line used in these experiments was a mouse/ mouse-hybridoma for production of monoclonal IgG1.
2.2. Culti6ation A bioreactor of 100-litre total volume (Biostat UD100, B. Braun Biotech International) with bubblefree aeration was used for the pilot scale cultivation of hybridoma cells. The growth medium contained the following proteins: human serum albumin (500 mg/l); human transferrin (10 mg/l); and bovine insulin (10 mg/1). A detailed description of the ingredients and the cultivation procedure is given elsewhere [16]. A single batch was used in the experiments.
2.3. Expanded bed adsorption process A Streamline 50 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for the studies (50 mm diameter; 1000 mm length). The experimental setup consisted of two Watson-Marlow Type X100 peristaltic pumps (Watson-Marlow Ltd, Falmouth, UK) with a 4.5-mm internal diameter Marprene synthetic rubber tubing. The UV absorbance for protein detection of the stream leaving the column was measured in a flow spectrometer (Knauer, Berlin, Germany) at 280 nm and was monitored by a X-Y-recorder (Kipp und Zonen, Solingen, Germany).
2.4. Cation exchange Streamline SP (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was filled into the column to a sedimented bed height of 33 cm (650 ml adsorbent volume). Equilibration was carried out for 50 min at 240 cm/h to result in a 2.5-fold bed expansion. The following buffers were employed: 1. Dilution buffer: 10 mM potassium phosphate-buffer (pH 5.0) 2. Equilibration buffer: 50 mM potassium phosphatebuffer (pH 5.5) 3. Wash buffer: 50 mM potassium phosphate-buffer (pH 5.5) 4. Elution buffer: 20 mM potassium phosphatebuffer+ 1 M NaCl (pH 8.0) The 20-litre harvest was diluted to 40 litre with dilution buffer and adjusted to pH 5.5 with 10% phosphoric acid. This was performed slowly with stirring to prevent local concentration peaks resulting in undesired cell damage. The cell concentration after dilution was 1.2×l06 cells per ml at 89% viability. The suspension was stirred at 80 rpm, applying a total volume of 32
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
litre to the column. During sample application, the flow was reduced from 240 cm/h to 180 cm/h in order to maintain the 2.5-fold bed expansion.
2.5. Affinity adsorbent Streamline rProtein A (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used at a bed height of 22.5 cm (450 ml adsorbent volume) in a Streamline 50 column. The fluid velocity was kept constant at 380 cm/h resulting in a bed expansion of 2.7-fold at the beginning and 2.9-fold at the end of the feeding phase. The following buffers were employed: 1. Equilibration: 50 mM potassium phosphate-buffer (pH 7.5) 2. Wash: 50 mM potassium phosphate-buffer (pH 7.0) Elution 100 mM sodium citrate-buffer (pH 4.0) The feedstock was slowly adjusted to pH 7.5 with a 10% sodium hydroxide solution and stirred in a tank at 60 rpm. The total feed volume amounted to 66 litres.
2.6. Analytical methods Off-line samples of the effluent and the feedstock were taken at intervals of 45 min during the feeding phase. The concentration of cells, intra- and extracellular LDH and extracellular DNA were analysed in all samples.
2.7. Cell concentration offline Cell concentration and viability in culture broth samples were determined by trypan blue dye exclusion (Fluke, Neu-Ulm, Germany) in a Neubauer haemocytometer.
2.8. Wash out of cells In order to determine hybridoma cell desorption from the saturated resin, the optical density of the effluent in the wash step was monitored online using a flow spectrometer (Knauer, Berlin, Germany) at 600 nm. The linear range of detection was up to a cell concentration of 2× 106 cells/ml.
2.9. Lactate dehydrogenase acti6ity The extent of cell lysis was determined by measuring lactate dehydrogenase (LDH) activity by reduction of pyruvate to lactate, which is coupled to the oxidation of NADH to NAD [17]. The reaction mixture contained PBS, pH 7.4, pyruvate, NADH, and depending on the sample 100–300 ml of test solution in a volume of l ml. Absorbance was monitored at 340 nm for 2 min at a scanning rate of 50 scans/min using a spectrophotometer (UVIKON 930, Kontron, Neufahrn, Germany).
161
LDH activity was calculated from the linear regression curve using the molar extinction coefficient of NADH of 6300 M − 1 cm − 1. Maximum LDH activity in the samples was obtained after disruption of cells by sonification.
2.10. DNA concentration DNA concentration was measured with an intercalating fluorescent dye (Pico Green P-7581, Molecular Probes Inc., USA). An aliquot of 100 ml of Pico Green was diluted in 20 ml, 10 mM Tris–HCL with 1 mM EDTA pH 7.5. From this solution, 500 ml was mixed with 500 ml of sample. After an incubation of 3 min in the dark the samples were measured in a fluorescence spectrofluorometer exited at 480 nm and the fluorescence intensity was measured at 520 nm. A calibration curve was obtained by measuring calf thymus DNA.
2.11. Product concentration MAb concentration was detected with a standard sandwich-ELISA assay as described by Ray et al. [18].
3. Results and discussion
3.1. Cell retention One of the main interaction problems between biomass present in the broth and the adsorbent is the retention of cells and/or the formation of cell-adsorbent aggregates during sample application. Analogous to protein adsorption, cell retention during sample application in an EBA process can be described by a breakthrough curve. The cell concentration in the effluent can be measured and plotted as c/co versus the normalized effluent time (U = t/t). If no interaction is present, cell breakthrough should coincide with the breakthrough of a tracer (e.g. acetone). This hydrodynamic breakthrough can be simulated by the dispersion model (eqn (1)) with the dimensionless Bodenstein number, Bo, describing axial mixing [19]. TypTable 1 Concentration and amount of DNA in the feedstock, effluent and eluate of Streamline SP and Streamline rProtein A EBA DNA
Feed Effluent Elution
Streamline r Protein A
Streamline SP
Mean DNA conc. (ng/ml)
Total DNA (mg)
Mean DNA conc. (ng/ml)
Total DNA (mg)
3748 3648 903
254.9 248.1 0.6
1032 744 14016
33.0 23.8 10.3
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
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Fig. 1. Normalized hybridoma cell concentration in the effluent of the cation exchange expanded bed adsorption during sample application. Determination of the experimental data (open triangle) by haemocytometer counting. The line represents a simulation of breakthrough under non-binding conditions using eqns (1) and (2), Bo = 60.
Fig. 2. Normalized hybridoma cell concentration in the effluent of rProtein A expanded bed adsorption during sample application. Determination of the experimental (open triangle) data by haemocytometer counting. The line represents a simulation of breakthrough under non-binding conditions using eqns (1) and (2), Bo = 60.
ical values for Bo in EBA systems of the configuration used here are in the order of magnitude of 60 – 80.
homogenization of the broth is necessary in order to release the product. A disruption of cells before or during thc adsorption step is undesirable because this would additionally contaminate the supernatant and hamper the efficiency of the subsequent purification steps. Furthermore, released substances may serve as mediators of cell adsorption to the stationary phase. Thus cell/adsorbent interactions always have to be examined in combination with potential cell damage. In these experiments, three parallel measures of culture viability were employed: trypan blue dye exclusion, LDH and DNA [20] release by the cells. These assay techniques were used to determine the amount of damage to the cells that has occurred during storage in the feedstock as well as by passing the pump and the expanded bed system. The release and uptake of the tracers is affected by membrane permeability and expresses the viability of cells. The three techniques were used in parallel because of the possibility that the experimental conditions may bias the assays. In contrast to LDH and DNA measurements, the haemocytometer technique does not allow the quantification of shredded cells as they can occur at high shear forces or by autolysis. The difference of molecular weight of the tracers (trypan blue Mr = 960.8, LDH Mr = 140 000) may also lead to a variance of results depending on the extent of membrane damage. Furthermore, the released DNA may interact with the adsorbents indicating an influence on fluidization stability. Differentiation between dead and total cell concentration is feasible by quantifying LDH activity in the supernatant as well as in the cell lysate. Fig. 3 presents the LDH activity during sample application to Streamline SP. A comparison of the supernatant data indicates
< < ' ==
c (1 − U) F = =0.5 1−erf c0 U 2 Bo U*L Bo= Daxl
(1)
(2)
The cell concentration in the effluent of the cation exchange adsorption is compared with the hydrodynamic breakthrough curve in Fig. 1. The area between these two curves represents the quantity of the retained hybridoma cells during the application within 7 – 8 residence times. Beyond this point almost the same concentration of cells can be found in the effluent as in the feed, so no additional retention takes place and the matrix apparently is saturated with biomass. Different results were obtained when hybridoma culture broth was applied to the affinity adsorbent as shown in Fig. 2. No cell retention was evident during sample application over the first 15 residence times. The decreasing cell concentration appearing in the effluent during the late feeding phase, was not caused by the adsorbent matrix. The long feeding time of 10 h for a 66-litre culture broth caused accumulation of cells at the lower distributor net (stainless steel, 70 mm mesh size) and cells were thus mechanically filtered out from the feed solution, as found by visual inspection after the experiment was completed.
3.2. Cell damage Hybridoma cells secrete IgG1 into the medium, no
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
Fig. 3. Activity of lactate dehydrogenase (LDH) in the feed and effluent of the cation exchange expanded bed adsorption process; (closed square) activity of the feedstock after cell disruption; (open square) activity of the effluent after cell disruption; (closed triangle) extracellular activity of the feedstock; (open triangle) extracellular activity of the effluent.
that no detectable damage to the cells during passage through the expanded bed system has occurred. Cell lysis, however, can be observed in the feedstock tank. Large shifts in pH and dilution prior to sample application have important effects on culture sensitivity to shear and autolysis and may be seen as the reason for a steadily decreasing viability of cells in the feedstock [15]. The total volumetric LDH activity after cell disruption was proportional to the total cell concentration. Thus the difference between LDH activity after cell disruption in the feed and the value in the effluent from the EBAcolumn is an additional measure of cell retention. The LDH data coincide satisfactorily with the haemocytometer results in Fig. 1. This implies that the determination of LDH activity in the supernatant and in the cell lysate is sufficient for both the analysis of cell retention, and detection of cell damage. It is important, however, that this protein is not adsorbed by the resin under the conditions of sample application. LDH measurements in the supernatant during adsorption to the rProtein A matrix revealed no detectable damage of the hybridoma cells during a single passage through the bed (Fig. 4). Major cell lysis was again observed in the feedstock tank. LDH activity increased after 20 column volumes had been applied. In this case, the long residence time of the culture broth in the feed tank without a supply of oxygen also negatively influenced cell viability.
3.3. DNA contamination The concentration of nucleic acids is of major concern
163
Fig. 4. Extracellular activity of LDH in Streamline rProtein A EBA; (triangle) feedstock; (square) effluent.
in pharmaceutical products, as the limit of DNA exposure to patients is l00 pg/dose per day [21]. If DNA is not bound to the adsorbent, its concentration can also be used to detect the rate of cell damage as described above. Thc graphs in Fig. 5 reveal DNA adsorption onto Streamline SP, especially during the first 30% of sample application. An analysis of the eluate shows a nearly 3-fold reduction in the total DNA load. About 15 mg DNA/ml adsorbent was bound and co-eluted during IgG1 desorption. Streamline rProtein A showed no detectable affinity for DNA and the overall DNA content of the sample containing monoclonal antibody could be reduced 290-fold during the EBA process (Table 1).
Fig. 5. Concentration of DNA in the supernatant during adsorption of MAb on Streamline rProtein A (triangle) and Streamline SP (square), open symbols refer to feedstock, closed symbols refer to effluent.
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
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Table 2 Concentration and amount of IgG1 in the feedstock, effluent and eluate of Streamline SP and Streamline rProtein A EBA MAB
Feed Effluent Elution
Streamline r Protein A
Streamline SP
Mean IgG1 conc. (mg/ml)
Total IgG1 (mg)
Mean IgG1 conc. (mg/ml)
Total IgG1 (mg)
50.0 14.0 3667.5
3400 954 2347
21.0 0.84 826.2
672.0 26.9 607.2
3.4. Product reco6ery A comparison of capture step efficiency of both matrices is summarized in Table 2. The breakthrough capacity of IgGl at 10% breakthrough (protein effluent concentration equal to 10% of the feed concentration) was 3.3 mg IgG1/ml Streamline rProtein A. In the experiment using Streamline SP the breakthrough of IgG1 did not exceed 2.5%. In both experiments, the yield of IgG1 was 95% and no IgG1 was found in the wash fraction. The elution volume for the affinity adsorbent was 1.42 bed volumes (sedimented), and 1.13 bed volumes for the cation exchange application.
3.5. Cell wash out The delayed cell breakthrough during EBA is either caused by mechanical retardation or by adsorption of biomass onto the surface of the adsorbent. In order to differentiate between these two phenomena, the effluent
Fig. 6. Online measurement of hybridoma cell concentration in the effluent during washing procedure of Streamline SP EBA; (thin line) 65% viability ; (intermediate line thickness) 0% viability ; (thick line) simulation under non-binding conditions using eqns (1) and (2), Bo= 60.
of the wash step was monitored with a flowthrough spectrophotometer. Optical density measurements at a wavelength of 600 nm are proportional to cell concentration of up to 2× 106 cells/ml and can give online information on cell clearance from the matrix during the washing procedure. In Fig. 6 the wash out behaviour from fluidized Streamline SP saturated with biomass of different viability is compared with the wash out function of a tracer under similar conditions as during sample application (Bol=60). The coincidence of the curves clearly demonstrates that the cells are adsorbed to the matrix and cannot simply be washed from the void volume of the bed.
4. Conclusions Expanded bed adsorption proved to be a gentle technique for the recovery of proteins from whole culture broth. This is of special interest for secreted products, as in the case of IgG1, when an additional cell lysis should be avoided in order to minimize contamination. The examined hybridoma cells did not exhibit any detectable cell damage. Taking into account that mammalian cells are one of the most shear sensitive organisms used in biotechnological production, it is a realistic assumption that neither bacteria nor yeast cells will experience damage caused by the passage of cells through the expanded bed or in the pumps used. Cell lysis resulting from unfavourable conditions in the feedstock tank can be reduced by minimizing the residence time of culture broth adjusted to feeding conditions. This could be achieved by inline dilution and pH adjustment in a relative small mixing tank situated between the cultivation reactor and the EBA system. A prerequisite for large scale applications is the reliability of the process. Biomass and DNA adsorption onto the resin beads is one of the major reasons for fluidization instability. Therefore it is of importance to quantify these parameters when investigating a new application for EBA. While adsorption of hybridoma cells was not detected with Streamline rProtein A and DNA adsorption was negligible, Streamline SP presented a distinct adsorption of cells onto the matrix that could not be desorbed in a conventional wash step. In addition, 16 mg DNA/ml Streamline SP was bound and co-eluted with the protein product. Cell adsorption to Streamline SP resins was also observed with other hybridoma cultures. The reason for this as well as the cause for DNA adsorption remains to be investigated. These aspects in combination with a satisfying breakthrough capacity of the affinity adsorbent suggest the use of Streamline rProtein A in an EBA mode as a
J. Feuser et al. / Process Biochemistry 34 (1999) 159–165
reliable initial antibodies.
purification
step
for
monoclonal
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