FPLC purification of mouse monoclonal antibodies from ascitic fluid using blue DEAE and thiophilic sorbents

103

Journal of Immunological Methods, 136 (1991) 103-109

© 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0022-1759/91/$03.50 ADONIS 002217599100064T JIM 05812

FPLC purification of mouse monoclonal antibodies from ascitic fluid using blue DEAE and thiophilic sorbents Erkki J u r o n e n 1, Jiiri Parik 1 a n d Peeter T o o m i k 2 I Institute of General and Molecular Pathology, Tartu University, 202400 Tartu, Veski 34, Estonia, U.S.S.R., and 2 ESTAR Biotechnology Development Company, Estonian Biocenter, 202400 Tartu, Mureli 16, Estonia, U.S.S.R.

(Received 8 January 1990, revised received 16 July 1990, accepted 4 October 1990)

Two fast methods for the purification of mouse monoclonal antibodies from ascites fluids using Blue-DEAE and 'thiophilic' adsorbent (T gel) in the FPLC system are described. Blue-DEAE chromatography is useful only for IgG1, IgG2a and IgG2b monoclonal antibody purification. T gel is a satisfactory adsorbent for IgG2b purification. Other IgG subclasses and IgM can also be obtained by simple one-step procedures, but the preparations contain small amount of high molecular weight contaminants. Key words: Chromatography, fast protein liquid; Monoclonal antibody purification; Blue DEAE gel; Thiophilic adsorption

Introduction

Pure antibodies are required for a number of scientific, diagnostic and therapeutic applications, and a wide spectrum of techniques are available for the purification of Mabs. The most popular methods are ammonium sulfate, caprylic acid precipitation, anion exchange chromatography, immunoaffinity chromatography, fractionation on protein A gels, etc. Unfortunately those methods are often time-consuming or limited by poor recovery.

Correspondence to: E. Juronen, Institute of General and Molecular Pathology, Tartu University, Veski 34, 202400 Tartu, Estonia, U.S.S.R. Abbreviations: Mab, monoclonal antibody; ELISA, enzyme-linked immunoadsorbent assay; HRP, horseradish peroxidase; PBS, 20 mM sodium phosphate buffer containing 0.15 M NaCI; PBS-T, PBS containing 0.05% Tween 20; PEG, polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

With recent advances in high performance liquid chromatography (HPLC) of proteins new methods for the purification of Mabs have become available (Jungbauer and Wenisch, 1989). However, due to the unique character of each antibody-producing clone (and the antibodies secreted), no universal purification method exists. Two separation routines are described, which have resuited in high purity Mabs in our experiments. The first method, based on chromatography on Cibacron Blue F3 GA-DEAE adsorbents, was first described by Bruck et al. in 1982. We have found that this method is only satisfactory for mouse IgG1, IgG2a and IgG2b antibodies. The second method, based on salt-promoted adsorption on thiophilic sorbent ('T gel'), was introduced by Porath et al. (1985) and subsequently used for Mab purification (Belew et al., 1987). Previously these methods have used only soft agarose gels but we have now succeeded in modifying both methods for use in the FPLC system.

104 All subclasses of mouse IgG and murine IgM have been purified using these techniques.

Materials and methods

Monoclonal antibodies and antigens All Mabs were developed in our institute. Ascitic fluid containing mouse Mab of IgG1 subclass reactive with human myoglobin were produced as described previously (Juronen et al., 1988). Ascitic fluid containing Mab of IgG2a subclass reactive with human neuron-specific enolase was kindly provided b y Dr. Jaanis Kasesalu. Ascitic fluids containing IgG2b and IgG3 Mabs reactive with progesterone and progesteroneovalbumin conjugate were provided by Dr. Andres Valdman. Ascitic fluid containing IgM specific for horseradish peroxidase (HRP) was provided by Dr. Rein Sikut. HRP was obtained from Sigma Chemical Co., St. Louis, MO. Adsorbents Cibacron Blue F3 GA (Serva, Heidelberg, F.R.G.) was coupled to TSK-gel DEAE-Toyopearl 650F (Toyo Soda, Tokyo, Japan) as described by Dean (1979) using 2 mg dye/ml of gel. All of the dye was bound under the coupling conditions used. The thiophilic adsorbent was prepared as follows: TSK-gel Toyopearl HW 65F (Toyo Soda, Tokyo, Japan) was washed with 1 M sodium carbonate buffer, pH 11.0 and suspended with 2 vols. of the same buffer. Divinyl sulfone (0.1 vol.) (Fluka, F.R.G.) was added and the mixture stirred for 1 h at room temperature. The gel was then filtered and washed with 0.5 M sodium carbonate buffer, pH 10.0 and suspended in 1 vol. of the same buffer. 0.1 volume of ]3-mercaptoethanol was added and the mixture stirred overnight. The final product was washed with distilled water. 5 vols. of buffer were used for every volume of gel during each washing step. Chromatography All chromatographic experiments were carried out using the Pharmacia-LKB standard FPLC system and HR 16/10 columns. Prior to chromatog-

raphy on Blue DEAE-gel ascites were clarified by centrifugation and immunoglobulins precipitated by the addition of an equal volume of saturated ammonium sulfate solution. Precipitated Mabs were recovered by centrifugation, dissolved in distilled water and dialysed overnight against 20 mM Tris-HC1 buffer, pH 7.2. The precipitate formed was removed by centrifugation and filtration through a 0.2 ~m filter. Omitting the precipitation step resulted in contamination of the IgG preparation with serum albumin. The sample, corresponding to 1 ml of ascites was applied to 10 ml blue DEAE gel at a flow rate of 1 ml/min. The column was eluted successively with 20 mM Tris-HC1, pH 7.2 (10 ml), and then with the same buffer containing 30 mM NaC1 (15 ml), 70 mM NaCI (15 ml) and 1 M NaC1 (15 ml). When T gel was used, the clarified ascites (1 ml) were mixed with an equal volume of 20% (w/v) ammonium sulfate in 0.1 M sodium phosphate buffer, pH 8.0, and filtered. The solution was then applied to the column (10 ml) which had been previously equilibrated with 0.1 M sodium phosphate, pH 8.0, containing 0.1 M NaCI and 10% (w/v) ammonium sulfate. The column was eluted with 20 ml of starting buffer, and antibodies were eluted by removing ammonium sulfate from the buffer. The column was regenerated with 95% ethanol after every five runs. All chromatography experiments were performed at room temperature, and 2 ml fractions were collected. Peak fractions were pooled and analysed by SDS-PAGE.

Indirect ELISA determination of antibody activity and quantity" 100 ~1 aliquots of antigen solution (1 btg/ml in PBS) were transferred to the wells of microtiter plates (Nunc, Copenhagen, Denmark) and incubated overnight. The plates were washed twice with PBS containing 0.05% Tween 20 (PBS-T) and then blocked with casein in PBS for 30 rain. Chromatographic fractions were diluted 1/1000 with PBS and 100 /xl aliquots added to the wells and incubated for 90 min. The plates were then washed three times with PBS-T and 100/~1 of goat anti-mouse Ig-HRP conjugate (affinity purified and conjugated in our laboratory) diluted 1/1000 in PBS-T were added. The plate was then in-

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106

cubated for 90 rain, washed four times with PBS-T and 100 /~1 of substrate solution (0.5 m g / m l ophenylenediamine and 0.03% hydrogen peroxide in 0.1 M citrate-phosphate buffer, pH 5.0) added. The color was allowed to develop in the dark for 30 rain. The reaction was stopped by adding 25/~1 of 12.5% sulfuric acid per well. Absorbance was measure using a Dynatech MR 580 MicroELISA A u t o R e a d e r ( D y n a t e c h Instruments, Santa Monica, CA) at 490 rim. For experiments with anti-HRP IgM a simpler procedure was used: the plates were coated with goat anti-mouse Ig antibodies (10 /~g/ml in PBS) overnight, washed, blocked and incubated with chromatographic fractions as above, 100 ~1 of HRP solution (1 /~g/ml in PBS) were added, the plates washed and absorbance read as described above after 30 min incubation. All procedures were carried out at room temperature. For the quantification of antibody in ascites and in purified material Nunc ELISA plates were coated overnight with 100 /zl of affinity purified goat anti-mouse Ig antibodies (free of cross-reactivity with goat IgG) at a concentration of 1 /~g/ml. The plates were washed with PBS-T and blocked for 30 min with casein-saturated PBS. 20 /~1 aliquots of the antibody solutions to be measured and standards diluted 1/40,000 plus 100/~1 of PBS-T containing PEG 6000 (PBS-T-PEG) were transferred to the wells of a microtiter plate and incubated 60 rain with shaking. The plates were washed three times with PBS-T and 100/~1 of goat anti-mouse Ig-HRP conjugate diluted in PBS-TPEG were added. The plates were incubated for 60 min with shaking, washed and enzyme reaction was performed as described above.

Results and discussion

Purification of Mabs on blue D E A E gel Typical elution patterns of mouse monoclonal IgG1, IgG2a and IgG2b are shown in Figs. 1A-1C. Three peaks were obtained, the first containing transferrin and negligible amount of IgG, the second containing pure lgG (Fig. 2) and the third containing all other ascitic proteins and some antibody activity. Using slightly different elution conditions (20 mM NaC1 for column washing and 30-50 mM NaC1 for IgG elution), Bruck et al. (1982) were unable to avoid contamination of IgG with transferrin. Thus the molarity of NaC1 in the elution buffers seems to be of crucial significance. Under our experimental conditions omitting the ammonium sulfate precipitation step leads to the contamination of lgG with albumin. Murine lgG3 apparently precipitated during the dialysis step and partially lost activity, since only inactive or degraded IgG3 was obtained by chromatography (Fig. 1D and Fig. 2, lanes 6-9). On the other hand, IgG3 prepared ammonium sulfate precipitation was still active. Similar data were obtained by 1

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SDS gel electrophoresis SDS-PAGE analysis of the pooled fractions was carried out under reducing conditions in 10% polyacrylamide gels according to the procedure of Laemmli (1970). After standard Coomassie staining the gels were additionally stained with silver ions according to the procedure of Damerval et al. (1987). The molecular weight markers trypsinogen (24 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), ovalbumin (45 kDa) and bovine serum albumin (66 kDa) were obtained from Sigma (St. Louis, MO).

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Fig. 2. Analysis by SDS-PAGE of Blue DEAE-Toyopearl chromatography peaks shown in Fig. 1. Lanes: 1, 10~ standards: 2. IgGl purification, peak 1; 3, IgGl purification, peak 2; 4, IgG2a purification, peak 2; 5, IgG2b purification, peak 2; 6, lgG3 purification, peak 1; 7, lgG3 purification, peak 2: 8, IgG3 purification, peak 3: 9, pelleted material obtained during dialysis of IgG3 enriched ascites.

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Fig. 3. Elution profiles and antibody activity distribution of 1 ml mouse IgG ascitic fluid on T gel. Flow rate: 1 m l / m i n . Buffer A: 0.1 M sodium phosphate buffer, pH 8.0+0.1 M NaCI and 10% ( w / v ) ammonium sulfate. Buffer B: buffer A without a m m o n i u m sulfate. Arrow indicates buffer A change to buffer B. Fraction volume: 2 ml. A: mouse lgGl; B: mouse lgG2a; C: mouse IgG2b; D: mouse IgG3.

108 Garcia-Gonzales et al. (1988), who used low ionic strength precipitation for the purification of mouse IgG3 and IgM. Over 60 experiments in our laboratory have shown that the other IgG subclasses do not lose activity during the ammonium sulphate precipitation and dialysis steps. This method is not appropriate for the purification of IgM, since IgM either precipitates on dialysis or binds irreversibly to the column under the conditions used. Nevertheless IgM has been successfully purified using agarose-immobilized Cibacron Blue F3 GA (Johnson et al., 1987). The major advantage of this purification schedule is the extreme stability of the IgG obtained no loss in activity was detected after several years of storage at - 2 0 ° C in 50% glycerol. The main drawbacks of the method are its somewhat limited

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applicability, the time-required and the lower recovery (66-70%) compared with the T gel method.

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Fig. 4. Elution profile o b t a i n e d after c h r o m a t o g r a p h y of 1 ml m o u s e IgM ascitic fluid on T gel. C h r o m a t o g r a p h y c o n d i t i o n s are the same as in Fig. 3.

High levels of recovery (76 87%) were noted for all T gel preparations of immunoglobulin. Typical elution patterns of mouse IgG and IgM are shown on Figs. 3 and 4. The chromatographic behavior of IgG1, IgG2a, IgG3 and IgM antibodies was very similar: the unadsorbed peak contained no antibody activity, and all antibody activity was eluted when ammonium sulfate was omitted from the elution buffer. The purified Mabs were free of transferrin and albumin, but contained small amounts of high molecular weight contaminants (Fig. 5). The behavior of IgG2b was totally different: it bound tightly to T gel and could only be eluted with 60% ethylene glycol, whereas high molecular weight impurities, which were present in other Ig subclass preparations, were eluted separately (Fig. 3C and Fig. 5). Similar results were obtained with

109 several different I g G 2 b clones (data n o t shown). However, Belew et al. (1987) reported that they were able to elute strongly b o u n d I g G 2 b from agarose-based T gel without using organic eluents. We have also tested T gels based on agarose a n d Spheron, b u t the results o b t a i n e d with Toyopearl were superior. O u r results suggest that T gel is the superior a d s o r b e n t for the p u r i f i c a t i o n of I g G 2 b since an extremely pure p r o d u c t c a n be o b t a i n e d at very low cost by a simple one-step procedure. The purity of other a n t i b o d y classes is also acceptable for most purposes, b u t if complete h o m o g e n e i t y is desired further p u r i f i c a t i o n b y gel filtration or b y c h r o m a t o g r a p h y o n Blue D E A E - g e l becomes necessary. The m a i n advantage of the m e t h o d s described here - the low price of the a d s o r b e n t c o m p a r e d with alternatives, e.g., i m m o b i l i z e d p r o t e i n A or p r o t e i n G, offers significant advantages in the case of large-scale preparative separations.

References Belew, J., Maisano, F. and Belew, M. (1987) A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a 'thiophilic' adsorbent. J. Immunol. Methods 102, 173.

Bruck, C., Portetelle, C., Glineur, C. and Bollen, A. (1982) One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel Blue chromatography. J. Immunol. Methods 53, 313. Damerval, C.~ Le Guilloux, M., Blaussonneau, J. and De Vienne, D. (1987) A simplification of Heukeshoven and Dernick's silver staining of proteins. Electrophoresis 8, 158. Dean, P.D.G. (1979) Protein purification using immobilized triazine dyes. J. Chromatogr. 165, 301. Garcia-Gonzales, M., Bettinger, S., Ott, S., Oliver, P., Kadouche, J. and Pouletty, P. (1988) Purification of murine lgG3 and IgM monoclonal antibodies by euglobulin precipitation. J. Immunol. Methods 111, 17. Johnson, E., Miribel, L., Arnaud, Ph. and Tsang, K.Y. (1987) Purification of IgM monoclonal antibody from murine ascitic fluid by a two-step column chromatography procedure. Immunol. Lett. 14, 159. Jungbauer, A. and Wenisch, E. (1989) High performance liquid chromatography and related methods in purification of antibodies. In: Monoclonal Antibodies: Production and Application. Alan R. Liss, New York, p. 161. Juronen, E.I., Viikmaa, M.H. and Mikelsaar, A.-V.N. (1988) Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies. J. Immunol. Methods 11l, 109. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteiophage T4. Nature 227, 680. Porath, J., Maisano, F. and Belew, M. (1985) Thiophilic adsorption - a new method for protein fractionation. FEBS Lett. 185, 306.