Feasibility Study for the Fractionation of the Major Human

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Anal. Chem. 2010, 82, 452–455

Feasibility Study for the Fractionation of the Major Human Immunoglobulin G Subclasses Using Hydrophobic Interaction Membrane Chromatography Lu Wang and Raja Ghosh* Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada Human immunoglobulin G (IgG) consists of four subclasses, each having specific biological functions and physical properties. Fractionation of these subclasses is challenging, both at analytical and preparative scales. This paper examines the feasibility of separating the major IgG subclasses, i.e., IgG1 and IgG2, by hydrophobic interaction membrane chromatography using environmentresponsive membranes. These were resolvable as separate peaks at appropriate eluting conditions. This method could potentially be developed further into a rapid and robust IgG subclass profiling technique, suitable for diagnostic applications. This method could also be used to produce specific subclass enriched IgG. Human serum immunoglobulin G (IgG) plays an important role in the immune system. There are four human IgG subclasses: IgG1, IgG2, IgG3, and IgG4.1 Members of a particular subclass generally respond to, recognize, and bind specific types of antigen.2-4 IgG1 and IgG2 are the two major subclasses accounting respectively for 43-75% and 16-48% of total serum IgG in healthy adults.5 During infections and immunologically compromised situations, these proportions change significantly.6,7 Women having healthy pregnancy have significant increases in their IgG1 levels during their first trimester when compared with nonpregnant women and women whose pregnancy ended in miscarriage.8 Thus, IgG subclass profiling of a patient’s serum is useful as a diagnostic tool. Quantitative analysis of human IgG subclasses is commonly carried out by ELISA. Reagents used in ELISA tests are very * Corresponding author. Phone: 905-525 9140ext. 27415. Fax: 905-521-1350. E-mail: [email protected]. (1) Terry, W. D.; Fahey, J. L. Science 1964, 146, 400–401. (2) Lal, R. B.; Buckner, C.; Khabbaz, R. F.; Kaplan, J. E.; Reyes, G.; Hadlock, K.; Lipka, J.; Foung, S. K. H.; Chan, L.; Coligan, J. E. Clin. Immunol. Immunopathol. 1993, 67, 40–49. (3) Scott, M. T.; Shackelford, P. G.; Briles, D. E.; Nahm, M. H. Diagn. Clin. Immunol. 1988, 5, 241–248. (4) Ottesen, E. A.; Skvaril, F.; Tripathy, S. P.; Poindexter, R. W.; Hussian, R. J. Immunol. 1985, 134, 2707–2712. (5) Hamilton, R. G. Clin. Chem. 1987, 33, 1707–1725. (6) Schur, P. H.; Borel, H.; Gelfand, E. W.; Alper, C. A.; Rosen, F. S. N. Engl. J. Med. 1970, 283, 631–634. (7) Musset, L.; Ghillani, P.; Lunel, F.; Cacoub, P.; Cresta, P.; Franguel, L.; Rosenheim, M.; Preud’homme, J. L. Immunol. Lett. 1997, 55, 41–45. (8) Wilson, R.; Maclean, M. A.; Jenkins, C.; Kinnane, D.; Mooney, J.; Walker, J. J. Fertil. Steril. 2001, 76, 915–917.

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expensive, and it frequently takes 2-7 h to complete a test.9,10 Other available techniques include radial immunodiffusion and rocket immunoelectrophoresis, which are time intensive, and nephelometry and turbidimetry which use expensive reagents.5 Moreover, sensitivity is critically dependent on the quality of the reagents used, i.e., the titer and purity of the antisubclass specific antibodies. Separation based analytical techniques such as chromatography, which relies on specific physical properties of the different subclasses, would be much more convenient and possibly more reproducible. Protein-A based affinity chromatography has been found to be suitable for isolating IgG3 from whole IgG.11,12 In a more recent study, the competitive binding of the different subclasses on protein-G has been discussed.13 However, the differences in binding were not significant enough to be exploited for subclass fractionation. Moreover, separation based on the differential binding on protein-A or protein-G would involve multiple steps with pH adjustments being required in between. Immunoaffinity chromatography based on immobilized polyclonal or monoclonal antisubclass specific antibodies has been used for subclass fractionation.5,12,14,15 This approach also involves multiple steps and is, therefore, complicated. Moreover, protein recovery in each step is low, typically in the 20-30% range.15 IgG4 has a lower isoelectric point than the other subclasses, and Skvaril and Morell16 exploited this to isolate it using multistep ion exchange chromatography. However, the major subclasses, i.e., IgG1 and IgG2, could not be fractionated using this approach. The molecular weights of IgG1, IgG2, and IgG4 are close to 146 kDa while IgG3 has a molecular weight around 170 kDa. IgG3 can, therefore, be distinguished from the other subclasses using electrophoretic methods.12 Other size based separation techniques such as size exclusion chromatography (SEC), ultracentrifugation, and field (9) Oxelius, V. A. Amer: J. Med. 1984, 76, 7–18. (10) Devey, M. E.; Bleasdale, K.; Lee, S.; Rath, S. J. Immunol. Methods 1988, 106, 119–125. (11) Kronvall, G.; Williams, R. C. J. Immunol. 1969, 103, 828–833. (12) Schauer, U.; Stemberg, F.; Rieger, C. H. L.; Borte, M.; Schubert, S.; Riedel, F.; Herz, U.; Renz, H.; Wick, M.; Carr-Smith, H. D.; Bradwell, A. R.; Herzog, W. Clin. Chem. 2003, 49, 1924–1929. (13) Viera, C.; Yang, H.; Etzel, M. R. Ind. Eng. Chem. Res. 2000, 39, 3356– 3363. (14) Bird, P.; Lowe, J.; Stokes, R. P.; Bird, A. G.; Ling, N. R.; Jefferis, R. J. Immunol. Methods 1984, 97, 97–105. (15) Persson, M. A. A. J. Immunol. Methods 1987, 98, 91–98. (16) Skvaril, F.; Morell, A. J. Immunol. 1970, 104, 1310–1312. 10.1021/ac902117f  2010 American Chemical Society Published on Web 12/10/2009

flow fractionation (FFF) could potentially be used to isolate IgG3, but the other subclasses would remain inseparable. Lederer and Bridonneau17 attempted to separate human IgG subclasses using thiophilic resin based column chromatography, but the eluted peaks were not sufficiently resolved. The main structural differences between the subclasses lie in the hinge region.5 While the heavy chain constant domains of any two subclasses share greater than 95% homology,18 the hinge regions are quite different in terms of the numbers and types of amino acids as well as the number of interchain disulfide bonds.5 In a recent study,19 it was demonstrated that the binding of IgG on hydrophobic surfaces took place through the hinge and CH2 domain. More recently, Yu and Ghosh20 using a totally different experimental approach from the earlier study,19 verified that the hinge and CH2 domain were involved in membrane binding. We, therefore, hypothesized that the structural differences in the hinge region of the different subclasses would affect their binding on hydrophobic surfaces and this could potentially be exploited to fractionate them. In previous studies, it has been shown that hydrophobic interaction membrane chromatography (HIMC) could be used for high resolution protein separation.21,22 This technique utilizes environment-responsive membranes with tunable surface hydrophobicity. The current study attempts to fractionate the major IgG subclasses, i.e., IgG1 and IgG2 using HIMC. Human IgG is first reversibly bound to a stack of environment-responsive membranes in the presence of an appropriate lyotropic salt followed by the elution of the subclasses in order of increasing hydrophobicity using a negative salt gradient. The fractionation of the major subclasses by HIMC is verified by ELISA. MATERIALS AND METHODS Human IgG (catalog no. I4506), standard human IgG1 (Kappa, catalog no. I5154), and standard human IgG2 (Kappa, catalog no. I5279) were purchased from Sigma Aldrich, Canada. Polyvinylidene fluoride (PVDF) microfiltration membrane (hydrophilized, 0.22 µm pore size, catalog no. GVWP 14250) used as an environment-responsive membrane for HIMC was purchased from Millipore, USA. Human IgG subclass determination ELISA kit (catalog no. 99-1000) was purchased from Invitrogen, Canada. All other chemicals used in the experiments, e.g., sodium phosphate (monobasic and dibasic), ammonium sulfate, and Bradford reagent were purchased from Sigma Aldrich, Canada. Solutions and buffers were prepared using ultrapure water (18.2 MΩcm) obtained from a diamond NANOpure water purification unit (Barnstead, USA). All samples and mobile phases used for HIMC were prepared in 20 mM sodium phosphate buffer (pH 7.0). Before use, all the buffers were microfiltered through a cellulose nitrate membrane (0.45 µm pore size) and degassed under vacuum. HIMC was carried out at ambient temperature (i.e., 24 °C) using a stack of six hydrophilized PVDF membrane discs (18 mm (17) Bridonneau, P.; Lederer, F. J. Chromatogr., B. 1993, 616, 197–204. (18) Coleman, P. M.; Deisenhoher, J.; Huber, R. J. Mol. Biol. 1976, 100, 257– 278. (19) Sun, X.; Yu, D.; Ghosh, R. J. Membr. Sci. 2009, 344, 165–171. (20) Yu, D.; Ghosh, R. Langmuir 2009, DOI: 10.1021/la902395v. (21) Ghosh, R. J. Chromatogr., A. 2001, 923, 59–64. (22) Wang, L.; Ghosh, R. J. Membr. Sci. 2008, 318, 311–316.

diameter) housed within a custom-designed membrane module23 which was integrated with an AKTAprime liquid chromatography system (GE Healthcare Biosciences, Canada). The UV absorbance (at 280 nm) and conductivity of the membrane module effluent and the backpressure were continuously recorded and logged into a computer using Prime View software (GE Healthcare Biosciences, Canada). The binding buffer contained 1.5 M ammonium sulfate; this was suitable for human IgG binding on the membrane stack while 20 mM sodium phosphate (pH 7.0) was used as eluting buffer. IgG feed solutions were prepared in binding buffer, and these were centrifuged at 10 000 rpm prior to injection. The HIMC was carried out at a 2 mL/min flow rate; the bound components were eluted out using an appropriate linear change in buffer composition. Samples corresponding to the eluted peaks were desalted using centrifugal ultrafilters (Amicon Ultra 30 kDa MWCO, catalog no. UFC903008, Millipore, USA) and analyzed by the Bradford assay for protein concentration. Samples (10 µL) were mixed with 200 µL of Bradford reagent (catalog # B6916, Sigma Aldrich, Canada) in 96 well plates. After a 5 min incubation, absorbance was measured at 595 nm using a microplate reader (680XR Bio-Rad, Canada). IgG subclass identity was confirmed using a slightly modified form of the ELISA protocol suggested by Invitrogen. One column of wells on a microplate was coated with monoclonal antihuman IgG1 while another was coated with monoclonal antihuman IgG2 followed by blocking with 1% ovalbumin solution for 30 min. The wells were emptied and washed three times. Samples corresponding to the HIMC peaks were then added to the wells in a row-wise fashion followed by 30 min of incubation. The wells were then emptied and washed three times. Peroxidase tagged antihuman IgG was then added to each well as the secondary antibody followed by 30 min of incubation. The wells were then emptied and washed, followed by addition of TMB (tetramethylbenzidine) substrate solution. The stopping reagent was added 10 min later, and absorbance was measured at 450 nm using a microplate reader. Human IgG was analyzed for the presence of aggregates by SEC with a Superdex 200 gel filtration column (10 mm i.d., 300 mm length, GE Life Sciences Canada, catalog no. 17-5175-01) using a HPLC system (Prepstar 218, Varian Canada). Sodium chloride solution (250 mM) was used as mobile phase at a flow rate of 0.2 mL/min. A 100 µL loop was used for sample injection. SEC experiments were also carried out using a high molecular weight gel filtration calibration kit (GE Life Sciences, Canada, catalog no. 28-4038-42). RESULTS AND DISCUSSION Figure 1 shows the chromatograms obtained with human IgG (Sigma catalog no. I4506) at two elution conditions: single step change from 0 to 100% eluting buffer (i.e., 0 mL gradient) and a 60 mL linear gradient. In each case, 5 mL of 0.15 mg/mL human IgG solution was injected into the membrane module. While a single eluted peak was obtained using a step change, human IgG could be resolved into several distinct peaks using the 60 mL gradient, indicating the presence of at least two major components and several minor components with subtle hydrophobicity differences. The human IgG used for obtaining the above chromatograms is known to be g95% pure (as determined by sodium (23) Ghosh, R.; Wong, T. J. Membr. Sci. 2006, 281, 532–540.

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Figure 1. HIMC chromatograms obtained with human IgG using a step change from binding to eluting buffer (0 mL gradient) and a linear gradient elution (60 mL) (number of membrane discs in stack: 6; sample volume: 5 mL; flow rate: 2 mL/min; eluting buffer: 20 mM sodium phosphate buffer, pH 7.0; binding buffer: eluting buffer + 1.5 M ammonium sulfate).

Figure 3. HIMC chromatograms obtained with standard human IgG1, standard human IgG2, a mixture of IgG1 and IgG2, and commercial human IgG (number of membrane discs in stack: 6; sample volume: 5 mL; flow rate: 2 mL/min; gradient length: 60 mL; eluting buffer: 20 mM sodium phosphate buffer, pH 7.0; binding buffer: eluting buffer + 1.5 M ammonium sulfate).

Figure 2. SEC chromatograms obtained with high molecular weight protein markers and human IgG.

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), and therefore, the occurrence of multiple peaks on the HIMC chromatogram would either indicate the presence of aggregates or could be due to the different subclasses. In a recent study,22 it was demonstrated that, with monoclonal antibodies, the tendency to interact with hydrophobic surfaces increase with degree of aggregation. In order to verify whether the multiple peaks on the HIMC chromatogram indicated the presence of aggregates, the human IgG sample was analyzed by SEC. The SEC chromatograms obtained with human IgG (see Figure 2) showed that it consisted primarily of IgG monomer (57.4 min retention time) 454

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and very small amounts of IgG dimer (49.5 min retention time). On the basis of these results, it may be presumed that the multiple peaks on the HIMC chromatogram obtained with 60 mL of linear gradient elution were due to the different IgG subclasses. Figure 3 shows the HIMC chromatograms obtained using 60 mL of linear gradient elution with four samples: (a) 5 mL of 0.1 mg/mL standard human IgG1, (b) 5 mL of 0.1 mg/mL standard human IgG2, (c) 5 mL of protein mixture containing 0.1 mg/mL IgG1 and 0.05 mg/mL IgG2, and (d) 5 mL of 0.15 mg/mL human IgG (Sigma catalog no. I4506). The injection of standard IgG1 resulted in a major peak having a retention time of 22.9 min while the injection of pure IgG2 resulted in a major peak having a retention time of 18.1 min. The presence of minor peaks in each of these chromatograms indicated the presence of small amounts of impurities. Since each of these samples is known to contain g95% IgG (as determined by SDS-PAGE), the impurities were presumably members of other subclasses or indeed aggregates. The mixture of IgG1 and IgG2 gave two major peaks, one corresponding to that obtained using standard IgG1 (ca. 23 min retention time), the other corresponding to that obtained using standard IgG2 (ca. 18 min retention time). The HIMC method could, therefore, separate IgG1 and IgG2 in ca. 30 min, this being much faster than ELISA, which typically takes about 2-7 h depending on the protocol in use. The samples corresponding to

Figure 4. Image of a portion of the ELISA plate used for determining identity of IgG subclasses in the major HIMC peaks. (Monoclonal antihuman IgG1 and antihuman IgG2 antibodies were used as primary antibody in columns 1 and 2, respectively, while the samples from major peaks 1 and 2 were tested in rows 1 and 2, respectively.)

the major HIMC peaks were analyzed by ELISA. Figure 4 shows a portion of an ELISA plate where monoclonal antihuman IgG1 and antihuman IgG2 were used as primary antibodies in columns 1 and 2, respectively. Samples from peak 1 were tested in the first row while samples from peak 2 were tested in the second. These results verify the presence of IgG1 in peak 2 and IgG2 in peak 1. The above results support the earlier observations19,20 that the hinge region plays a significant role in the interaction of IgG with hydrophobic surfaces. The IgG1 hinge is made up of 15 amino acid residues while that of IgG2 contains 12 amino acids.5 The number and composition of amino acids plays an important role in hydrophobic interaction.24 Moreover, IgG being a Y-shaped molecule, the interaction of the hinge region with a surface could potentially be influenced by the flexibility of the molecule. The rank order (most to least flexible) for the hinge-folding mode for the human IgG subclasses is IgG3 > IgGl > IgG4 > IgG2.25 The hinge region of IgG1 would, therefore, face less steric hindrance than the hinge region of IgG2 while interacting with surfaces. This could be a plausible explanation for the stronger interaction of IgG1 with the membrane. The current work was carried out as a feasibility study for fractionating IgG1 and IgG2 from commercial IgG using HIMC. IgG is commercially purified using techniques such as affinity and ion exchange chromatography. Earlier workers have shown that these techniques discriminate between the different subclasses.13,16 (24) Mitaku, S.; Hirokawa, T.; Tsuji, T. Bioinformatics 2002, 18, 608–616. (25) Roux, K. H.; Strelets, L.; Michaelsen, T. E. J. Immunol. 1997, 159, 3372– 3382.

The subclass composition of human IgG used in our study could, therefore, be significantly different from that in whole serum. Using appropriate calibration, the HIMC technique could be used to determine relative and absolute IgG1 and IgG2 content in a human serum. Future work will assess the feasibility of extending the capability of the current technique for fractionating the remaining subclasses, i.e., IgG3 and IgG4. If successful, this would form the basis for developing rapid and robust IgG subclass profiling techniques for diagnostic applications. As discussed earlier, specific types of antigens are known to trigger synthesis of specific IgG subclasses.6-8 Polyclonal antibody preparations used for neutralizing infections could, therefore, be made more specific and potent by enriching specific subclasses using HIMC, e.g., IgG2 enrichment in bacteria neutralizing polyclonal antibodies. CONCLUSIONS An environment-responsive membrane with tunable surface hydrophobicity could be used to separate IgG1 and IgG2 under appropriate eluting conditions. Human IgG1 interacts more strongly with a hydrophobic membrane surface than human IgG2, presumably due to the significant differences between the hinge regions of these major human IgG subclasses. The flexibility of the molecule could also potentially contribute toward a difference in binding. The HIMC technique in its current form is suitable for rapid profiling of the major human IgG subclasses. It could potentially be developed further for complete profiling of all four subclasses. The technique could be used for producing specific subclass enriched IgG fractions suitable for binding specific types of antigens. ACKNOWLEDGMENT We thank the Natural Science and Engineering Research Council (NSERC) of Canada for funding this study. Paul Gatt of the Department of Chemical Engineering, McMaster University is thanked for fabricating the membrane modules used in this study based on designs provided by R.G. L.W. thanks Shell Canada and China National Scholarship Council for personal scholarships. R.G. holds the Canada Research Chair in Bioseparations Engineering. Received for review September 21, 2009. Accepted November 7, 2009. AC902117F

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