Bull Vet Inst Pulawy 48, 321-327, 2004

ISOLATION AND PURIFICATION OF POLYCLONAL IgG ANTIBODIES FROM BOVINE SERUM BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
JAN STEC, LEOKADIA BICKA AND JACEK KUMAK
Department of Biochemistry, National Veterinary Research Institute 24-100 Puawy, Poland e-mail: stec@piwet.pulawy.pl Received for publication March 08, 2004.

Abstract
Three different high-performance liquid chromatographic (HPLC) techniques, i.e. affinity, ion exchange, and gel filtration chromatography, have been used to purify polyclonal antibodies from bovine serum. Polyclonal antibodies were obtained from animals infected with bovine leukaemia virus, which was confirmed by AGID and ELISA tests. Precipitation of the antibodies by ammonium sulphate prior to HPLC made possible to purify the antibodies in one chromatographic step. However, if the highest purity is required and separation IgG1 from IgG2, it should be used one and/or two additional columns. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the purest preparation revealed only one band without tailing or any additional extra peaks. Attempts were also made to purify the antibodies without prior ammonium sulphate precipitation. But the best results in immunoglobulins preparation were obtained in a combination of affinity, ion-exchange and gel filtration chromatography, and sample preparation by ammonium sulphate precipitation and delipidation by n-hexane. These preparations are comparable in purity to those commercially available immunoglobulin standards. The rapid HPLC techniques were found to be very useful for the purification of polyclonal antibodies on a preparative scale, where sample loading of up to 25 mg of serum protein could be fully resolved in satisfactory yields.

Key words: cows, serum, IgG, HPLC,

purification.

Purification of immunoglobulins (Ig) is required for many applications in numerous field of science and technology. A growing list of purification procedures has emerged, reflecting the heterogens nature of this group of molecules and also different researchers, demands for varying levels of purity. Most papers deal with monoclonal antibodies from mouse ascites as a starting material (3, 4, 7), and for the isolation of murine monoclonal IgG antibodies, affinity chromatography on immobilized protein A or G is probably the most commonly used technique (11). However, considerable

attention has been paid to alternative methods, for instance, when a special procedure is required in field of veterinary or human medicine (6). General adsorption technique, using high performance liquid chromatography (HPLC), is attractive in this respect. Recent works have shown that high performance ion exchange and high performance size exclusion have the potential for rapid isolation and/or purification of monoclonal and polyclonal antibodies from different range of biological material (2, 5). The classical protocols for the isolation and purification of IgG antibodies were frequently long and tedious, and often did not result in a high degree of the purity. For example, the purification of antibodies by conventional precipitation with ammonium sulphate followed by DEAE-cellulose chromatography which has been used to isolate polyclonal and monoclonal antibodies from mouse ascites fluid and animal serum requires several hours to complete and is often limited by poor recovery (11). Affinity chromatography using protein A has been successfully employed for the purification of mouse and human IgG, but one subclass (IgG1) was bound only weakly, and another ( IgG3) did not bound at all (12). Hydroxyapatite adsorption chromatography is very useful for simple and rapid fractionation of proteins, but has intrinsic limitations for routine use, and is difficult to scale-up for IgG purification from serum of animal species (8, 9). Some authors have demonstrated that IgG from human serum could be partially purified on an anion-exchange Mono Q column followed by gel filtration on Superose 6 column (6, 10). Recently, a mixed mode ion-exchange chromatography matrix, utilizing silica gel as the support, was used for the rapid purification of immunoglobulins. This chromatographic matrix demonstrated little or no affinity for albumin, transferrins, proteases or pH indicator dyes from tissue culture media (7). Separation and recovery of proteins from ionexchange chromatographic media are affected by factors such as buffer type and pH, length of gradient, flow rate of the mobile phase, ionic strength and nature of counter

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ion, and characteristic of the proteins (13). The selection of ideal conditions for protein purification involves changing some or all of these parameters. Recent development of high-performance liquid chromatography (HPLC) and fast performance liquid chromatography (FPLC), using combination of several columns, anion exchange, immunoaffinity, size exclusion, gel filtration and hydrophobic interaction, have opened up new possibilities for separation of polyclonal immunoglobulins from animal sera. In the present study, we have performed a comprehensive evaluation of high performance anion and cation exchange chromatography, immunoaffinity chromatography, and high performance size exclusion separation as a single-step technique or combine protocol for purification of polyclonal IgG antibodies from bovine serum. The samples were pretreated similarly to allow a direct comparison between different techniques. Ammonium sulphate precipitation and nhexane delipidation of serum prior to chromatography separation in relation to the final purity of polyclonal antibodies were also examined. Based on these findings, we propose a strategy for the purification of polyclonal IgG antibodies from bovine serum by high performance adsorption chromatography techniques.

Material and Methods

Samples. Blood samples were collected from cows infected with bovine leukaemia virus (BLV) by jugular venipuncture and serum was prepared in the usual manner, clarified by centrifugation (1000 g, 15 min) and prior to preparation of IgG diluted 1:1 with sodium phosphtate buffer at pH 7.2. The presence of polyclonal antibodies (pAb) against BLV was confirmed serologically by AGID and ELISA. Ammonium sulphate precipitation. The precipitation was performed at 40C. Equal volumes of diluted serum and saturated ammonium sulphate were mixed by slow addition of the ammonium sulphate solution during gentle stirring. On the next day this material was centrifuged (10 000 g for 20 min) and washed twice with 50% saturated ammonium sulphate solution. The precipitate was dissolved in distilled water and dialyzed against phosphate buffer (10 mM Naphosphate + 0.15 M NaCl, pH 7.2). The precipitate solution was mixed with equal volume of n-hexane and centrifuged (20 000 g, 40C for 30 min) to remove lipids. The final solution was filtered through a Millipore filter (0.22 m), and clear supernatant was loaded into the column. Instruments. Whole experiment was performed on HPLC instrumentation Akta Explorer 10 S (Pharmacia Biotech, Uppsala, Sweden) consisting of a solvent pump system model 900 and 910, gradient module M-925, detection system UV 280 nm, pH/C-900 monitor, fraction collector Frac-900, recorder HP-890. The HPLC system was controlled by software Unicorn ver. 3.0 with a Compaq computer. The fractions were

collected every 0.5 - 2.0 min in volume program mode, depending on the used method. Ion exchange chromatography. Ion exchange chromatography was performed on a Mono Q HR 5/5 anion exchange column (50 mm x 5 mm I.D., Phamacia Biotech, Uppsala, Sweden) with starting buffer A, 20 mM bisTris propane (pH 9.5), and final buffer B, 20 mM bisTris propane containing 1 M NaCl (pH 7.5). The gradient was generated over 20 min at a flow rate of 1 ml/min. The sample load was between 0.5 10 mg protein injected with 0.1 2.0 ml sample loop. Affinity chromatography. Affinity chromatography was performed on a Protein G Sepharose 4 (fast flow) 1 and 5 ml columns (Pharmacia). Equilibration buffer was 0.02 M Naphosphate, pH 7.2, and eluting buffer was 0.1 M glycine at pH 2.5. The column was washed with 0.1 M glycine buffer at pH 2.0, following immediately with equilibration buffer. The flow-rate used, at all stages was 1 ml/min. Samples were applied on the column by sample loop: 0.5 ml or 2.0 ml, and protein load was between 0.1 25.0 mg for analytical and preparative scale, respectively. The collected fraction of 1 ml volume were immediately treated with neutralizing 1 M Tris-HCl at pH 9.0. Gel filtration. Gel filtration was carried out on Superdex 200 Prep grade 10 x 300 columns (Pharmacia). The column was equilibrated with 0.05 M Na-phosphate buffer + 0.015 M NaCl, adjusted to pH 7.4. The samples (sample volume as rated in the manual from the column supplier) were subjected to chromatography at flow-rates of 0.5 ml/min. In order to determine the molecular weight of all sample components, a molecular weight calibration curve was set up. Buffer exchange. Buffer exchange or desalting of collected fractions, were carried out on a Fast Desalting HR 10/10 column packed with Sephadex G 25 Fine (Pharmacia). The samples were applied in 1.0 ml volume by sample loop and subjected to chromatography with buffer, into which the sample was to be transferred, at a flow-rate of 2 ml/min. SDS-PAGE electrophoresis. The purity of various IgG preparations was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of reduced and non reduced samples. The total polyacrylamide concentration in the separating gel was 8 or 12%. Coomassie brilliant blue R-250 was used to visualize the protein bands. As a standard, the lowmolecular-weight (LMW) references from MBI Fermentas (Vilnus, Lithuania) was used: -galactosidase MW 116 000; albumin MW 66 000; ovoalbumin MW 45 000; lactate dehydrogenase MW 35 000; restriction endonuclease Mw 25 000; -lactoglobulin MW 16 000; lysozyme MW 14 000. All samples and buffers used in this study were filtered through 0.22 m Millipore filters to remove small particles, and buffers were additionally degassed prior to use. After ten chromatographic separations on each column the clean in place (CIP) was carried out, according to Pharmacia protocols, to prevent loss in performance of the columns. Protein determination was

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performed by the Bradford method (1), using bovine serum albumin as a standard.

Results
In the first set of the experiments, bovine serum containing pAb against bovine leukaemia virus was subjected to chromatography. Before chromatography, ammonium sulphate precipitation and n-hexane delipidation was used as a general purification step. The result for the three consecutive columns are shown in Figs. 1 A, B, and 2 C. The main peak of IgG, after ammonium sulphate precipitation, was exclusively isolated by affinity chromatography on Protein G Sepharose column with a 0.1 M glycine eluting buffer at pH 2.5, but the remaining ballast proteins were removed with Na-phosphate starting buffer (Fig. 1 A). The protein G is covalently bound to Sepharose, and is able to trap IgG subclasses eg. IgG1, IgG2 and IgG3. The most of the ballast proteins were eliminated in the flow of starting Na-phosphate buffer at pH 7.2. Separation of IgG fractions to the subclasses was not successful on Protein G Sepharose column with stepwise elution gradient of pH ranged from 3.5 to 2.2 and cannot be recommended. When the purity of IgG fraction is not very important, the ammonium sulphate precipitation and protein G affinity chromatography can be used for processing of the isolation and purification of bovine polyclonal antibodies. The affinity Protein G column served very well on analytical scale with the protein load less than 0.5 mg and also can satisfactory work on small-preparative scale with the protein loading on the column up to 25 mg (Fig. 2 D). The chromatographic profile of this separation gave very broad and flattened peaks, however, the both peaks, the ballast protein and IgG fraction were separated very well. The second step of the purification of bovine pAb consisted in chromatography by anion exchange technique on Mono Q HR 5/5 column. This column was used for separation of the IgG peak obtained from the affinity column. The best results were obtained when elution of the proteins was performed with 20 mM bisTris propane buffer at pH starting from 9.5 and ending with the 7.5, applied with the linear gradient of the salt from 0 to 1 M NaCl concentration over 20 min at a flow rate of 1 ml/min. The resulting chromatogram has been shown on Fig. 1 B. The elution profile gave at least three main peaks. The first one was unidentified proteins as the remaining portion of ballast materials and this amount of waste proteins were removed in void volume of the column. The next two peaks had retention volume of buffer: 7.62 ml and 12.46 ml, and they completely distinguished one from another. According to retention volume of standard immunoglobulins, those two peaks were identified as IgG1 and IgG2. In a similar experiment, commercially available pAb from bovine serum was tested and the obtained results of affinity chromatography and anion exchange chromatography were very close to those from the previous preparation.

When a diluted bovine serum was run by ion exchange technique (Fig. 3 E) on Mono Q HR 5/5 column, the peak of IgG has been separated. According to the chromatogram profile, there were many different proteins accompanying the IgG fraction. The identity of IgG peak was confirmed by the standard sample of bovine IgG. However, the precipitation step removed more than 50% of serum proteins, the majority of proteins were the ballast proteins, among those the peak of IgG made up only few percent of the total protein content. The anion exchange column, Mono Q HR 5/5 was also used as the first column for the isolation of IgG fractions from ammonium sulphate precipitation. The results were satisfactory and there were obtained two well separated peaks identified as IgG1 and IgG2. However, the Mono Q HR column performance was disturbed after the several consecutive runs of ammonium sulphate preparation, and the routine cleaning up procedure was necessary to restore efficiency of the column. After cleaning in place (CIP), as recommended by the manufacturer, the column was tested for efficiency, to check column performance, by a standard mixture of two proteins: -lactoglobulin and ovoalbumin at concentration of 2 mg/ml each (result not shown). Even so, as the column was used for the next series of chromatographic runs, the live time of the columns have been shortened, and they should be withdrawn, when the cleaning procedure did not restore their performance. Because of high price of a Mono Q column and reasonably lower an affinity column, this was in support to used sequence of the two columns, the first Protein G Sepharose followed by Mono Q. Gel filtration chromatography on HP Superdex 200, 10/300 column was used as a final polishing step to obtain IgG1 and IgG2 fractions of very high purity. The column was loaded with 1 to 5 mg per ml of the individual IgG subfractions or both together and the best performance was achieved at a flow rate of 0.5 ml/min. This step allowed to cut of the immunoglobulin with molecular weight (MW) lower than 150 000 kD from any other proteins, or the immunoglobulins partially degraded during the all protocol of preparation. The chromatogram profile of the fine purity IgG1 fraction on the Superdex column is presented on Fig 2 C. The large peak represents IgG1 fractions (retention volume 11.52 ml) separated from the small one (retention volume 10.79 ml) which contained the rest of impurities. The purity of the various chromatographic fractions was analysed by SDS-PAGE (Fig. 4). Lane a-1 contains the pooled fractions from Fig. 1-A. There are present some protein tailing bands from the bovine serum. The peaks eluted from Mono Q column (Lane a-2) or Mono Q and Superdex 200 (Lane a-3) show the highest purity. Lane MW contains molecular weight marker proteins. SDS PAGE with reducing condition is presented on Fig. 4-b. The appropriate lanes: 5, 6, 7, and 8 correspond to the same samples that in Fig. 4-a, 1, 2, 3, and 4, respectively. On this figure, both subunits show molecular weights about 50 kD, which correspond to subunits of bovine IgG with broken - S - S - bonds.

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mAU

pH 7

mAU 1600 1200 800 400 0 0 10

waste IgG1

mS/cm

300
IgG

6 5

80
IgG2

200 100 0 0 5 10 15 ml
waste

60 40 20

4 3 2

15

20

25

ml

Fig. 1. A - Isolation of bovine IgG on Protein G Sepharose column. Low protein load (0.5 mg). Elution with 0.1 M glycine buffer at pH 2.5. B - Chromatographic pattern for bovine polyclonal antibodies obtained on Mono Q HR column with 20 mM bis Tris propane buffer (pH 9.57.5) and (01 M) NaCl gradient elution.

C mAU 800 600 400 200 0 0 10 20 30 ml

D mAU 4000 3000 2000 1000 0 0 5 10 15 ml

pH 7

waste IgG

6 5 4 3 2

Fig. 2. C - Gel filtration chromatography of pAb on Superdex 200 HR column. The large peak represents IgG1. Sample load of 1 mg protein. Elution with 50 mM sodium phosphate buffer at pH 7.2. D - Purification of bovine IgG on Protein G Sepharose column. High protein load (25 mg). Elution with 0.1 M glycine buffer at pH 2.5.

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E mAU 500 400 300 200 100 0 10 20 30

IgG

mS/cm 80 60 40 20 10 ml
0

F mAU 2500 2000 1500 1000 500

mS/cm 5.0 4.0 3.0 2.0 1.0

10 15

ml

Fig. 3. E - Chromatographic separation of diluted bovine serum obtained on Mono Q HR 5/5 column. Sample load was 2 mg protein. The elution with 20 mM bisTris propane buffer at 9.57.5 pH. The gradient of 1 M NaCl 0100% was generated over 20 min. F - Chromatograpic pattern of buffer exchange on Sephadex G 25 Fine 10/10 column. The sample load was 25 mg of protein after affinity chromatography. Isocratic elution with 20 mM bis-Tris propane buffer at the flow rate of 2 ml/min.

a
MW 116 kD 66 45 35 25 18 45 35 1 2 3 4 116 kD 66 MW 5

b
6 7 8

Fig. 4. SDS-PAGE non-reduced (a) and 2-mercaptoethanol-reduced (b) samples from various purification steps. MW - molecular weight reference, a-1 sample used in Fig. 1 A (affinity), a-2 sample used in Fig. 1 B (anion exchange), a-3 sample used in Fig. 2 B (gel filtration), a-4 sample used in Fig. 3 F (buffer exchange). Samples on gel b correspond to samples from gel a.

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The analysis confirmed the high purity of IgG1 and IgG2 fractions obtained by the three steps purification procedure. From these results it is obvious that the ammonium sulphate step gives a final pAb preparation of higher purity than that obtained if it is omitted. The yield of pAb after two step procedure (ammonium sulphate precipitation and Protein G Sepharose chromatography) was found to be about 75%. From various experiments with the Mono Q column followed by Superdex 200, the protein recoveries in our experiments were estimated to be about 50%. During all experiments, samples to be loaded into columns were applied in a starting buffer. The buffer exchange and/or desalting after gradient was performed by gel filtration chromatography on Sephadex G 25 Fine column (Fast Desalting HR 10/10). The flow rate of 2 ml/min and collected fractions of 1 ml allowed for completely separation of a protein peak from salt of a previous buffer. As an example, the typical chromatogram profile of buffer exchange is showed on Fig. 3-F. For example, the fraction of IgG, after affinity column step on Protein G Sepharose, in 0.1 M glycine buffer pH 2.5 neutralized with 1 M TrisHCL to pH 7, was separated from IgG fraction, removed and changed with 20 mM bis-Tris propane, pH 9.5 as the starting buffer for the Mono Q HR column. The protein peak was well separated from the salt peak exactly at the bottom of both peaks, and only such separation was acceptable.

Discussion
In this study, we have obtained highly purified polyclonal IgG antibodies, of G1 and G2 subclasses from bovine serum in combination of affinity, ion-exchange and gel filtration chromatography using HPLC system. While the present work deals with small scale purification, both the affinity and ion exchange used here can easily be scaled up. Affinity chromatography with Protein G Sepharose at pH 2.5 was shown to be a powerful method for the isolation and purification of IgG antibodies from bovine serum. This method is attractive since mild condition is used and, furthermore, since several of the proteins normally found in serum do not bind to the column. The capacity of the immunoaffinity bead is thereby used for binding the protein of interest. Generally, immunoaffinity column with protein G appears to be particularly suited for the isolation of IgG molecules from bovine serum. The affinity chromatography on Protein A Sepharose did not bind strongly bovine IgG and the performed experiment showed high losses of IgG which could not be accepted. However, this column is routinely used for the isolation and purificaton of IgG fractions from human and some

animal species sera: mouse, rabbit, guinea pig, dog, pig (9). By anion exchange with Mono Q column at pH 7.5 9.5, two IgG antibodies (subclasses IgG1 and IgG2) with isoelectric points above neutral, were purified almost to homogeneity. The high pH of starting buffer appears not to affect the immunoglobulin molecules. Similar results were obtained by anion exchange with Mono Q in other studies (11). The capacity of this technique is potentially lower than that of affinity with protein G when the bovine serum precipitate is used as a sample, since essentially all components in this fluid are adsorbed to the column. Furthermore, extremely tight binding of certain molecules may cause an increase in column backpressure which necessitates extensive cleaning procedure between experiments. However, pretreatment of the sample by ammonium sulphate precipitation or a similar method, may eliminate this problems (12). Such pretreatment may be particularly relevant when large sample volumes are used. Attempts were also made to purify polyclonal antibodies by high performance gel filtration on a Superdex 200 column. Evidently immunoglobulins generally have the molecular weight 150 kD compared to the other proteins in serum. This technique has been successfully used for the separation of IgG from ascites fluid and serum (5). The IgG fractions, which we obtained by additional purification on gel filtration using very fine molecular sieving column, were completely resolved from the proteins with molecular weight other than 150 kD. The antibodies used in this study were purified to near homogeneity in a combine method by these techniques. In conclusion, the following strategy for combine HPLC purification of bovine IgG antibodies from serum can tentatively be suggested, in cases when other columns are considered less suitable: (i) affinity chromatography on immobilized protein G at pH 2.5 is the first choice for antibodies due to mild condition employed; (ii) anion exchange chromatography for antibodies with high isoelectric points (>7.2), as the second column for further purification and separation of IgG1 from IgG2: (iii) gel filtration as the final polishing step to obtain very pure individual classes of IgG antibodies. If very high purification is not necessary, the ammonium sulphate precipitation and affinity chromatography on immobilized protein G should be satisfactory, but for obtaining a completely homogenous product, at least the second chromatographic step should be included. The last column optionally may be used for very fine purification of obtained bovine IgG subclasses. However, when ammonium sulphate precipitation must be avoided, diluted bovine serum can be subjected to chromatography directly on the anion exchange Mono Q column 5/5, as a starting material.

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Our results show that the HPLC techniques are efficient for the purification of pAb and that the analytical columns used in this study can be utilized for preparative scale purification. The use of larger columns will, of course, improve the capacity.

7.

8.

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