- Email: [email protected]
Mechanistic model for adsorption of immunoglobulin on hydroxyapatite
Journal of Chromatography A, 1142 (2007) 13–18
Mechanistic model for adsorption of immunoglobulin on hydroxyapatite Paul K. Ng a,∗ , Jie He a , Pete Gagnon b a
Process Applications, Process Chromatography Division, Life Sciences Group, Bio-Rad Laboratories, Hercules, CA 94547, USA b Validated Biosystems, San Clemente, CA 92672, USA Available online 11 December 2006
Abstract A model is developed for correlating the adsorption of protein to hydroxyapatite in terms of pH, phosphate and imidazole. It provides a basis for manipulating the chromatography conditions so as to obtain the best possible fractionation. The adsorption mechanism is apparently a function of two calcium affinity interactions and cation exchange. We used immunoglobulins and a fusion protein with or without a poly(6)histidine tag for our investigation. The results are presented and discussed in the present study. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxyapatite; IgG; Adsorption; Chromatography; Phosphate; pH; Resolution
1. Introduction Hydroxyapatite is a well-known multimodal chromatography support for purification of proteins and other biomolecules. While its multimodality and crystal structure are frequently credited for its unique selectivity, it also complicates screening and often leaves process developers at a loss with respect to optimizing a separation. Previous work with proteins such as IgG discussed retention on hydroxyapatite by a combination of two mechanisms: phosphoryl cation exchange which involves the interaction of positively charged amino groups on proteins with the P sites on the matrix; and calcium metal affinity which involves the protein carboxyl groups and the C sites on the matrix [1–4]. Using crystallography, Kawasaki [5] determined that the P sites and C cites are constructed from a single or several phosphate ions and a single or several crystal calcium ions, respectively. A third mechanism that has not been explored previously is metal–histidine interaction. Calcium ions can act as electrophiles, seeking to participate in electron pairs with other atoms so that a bond or charge–charge interaction can be formed. Such an interaction has been demonstrated between metal ions and the imidazole groups of histidyl side chains in a variety of proteins [6,7]. And in addition, affinity reten-
∗
Corresponding author. Tel.: +1 510 741 4839. E-mail address: paul [email protected] (P.K. Ng).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.071
tion of proteins by immobilized metal ions increased with the number of histidine residues [8,9]. Extension of these observations to IgG which carries multiple histidine residues on its complementarity-determining region (CDR) and the FC region suggests the possibility of histidine–calcium interaction as contributory to IgG retention. The present study attempts to determine the reactive constituents of the three mechanisms: phosphoryl cation exchange, carboxyl chelation and calcium–histidine interaction through the imidazole side chain of histidine residues. Results will be presented demonstrating the effect of pH, phosphate and imidazole on purification performance. Furthermore, this study applies statistical design to address the question, ‘what are the conditions for pH, phosphate and imidazole that optimize resolution?’. Statistical factorial design (SFD) provides the minimum number of experiments needed to evaluate the simultaneous effects of multiple variables and multi-variable interactions on predetermined processing objective [10,11]. Four stages are usually employed to apply SFD. First, ‘n’ variables that have an effect on process outcomes are selected. In our case these are pH, phosphate and imidazole. Second, anticipated ranges for each variable are identified for 2n experiments. Third, the process objectives are measured for each of the 2n experiments that span the variable ranges. Fourth, the effects of each variable and multi-variable interaction on predetermined process objectives are ascertained by commercially available Design of Experiments (DOE) software.
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P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
An important aspect of the present study is to facilitate investigation into purification performance of CHT chromatography as a tool to ensure elimination of aggregate in the purification of monoclonal antibody. Aggregation is one of the most important issues in the purification of monoclonal antibodies which are typically captured by protein A as the first chromatography step in a process train. Even with changes in the solvent conditions, such as adding surfactants, chaotropic agents and/or reducing agents or adjustment of wash conditions, presence of aggregates in the protein A eluate can still present significant technical challenge. These aggregates are known to be covalent or non-covalent interaction of denatured molecules. They can cause a loss of activity, reduced therapeutic value and untoward clinical effect [12,13]. It is therefore important to define process step to ensure its removal. Method such as size exclusion chromatography has been evaluated and was found to be laborious [14,15] because of low capacity and long chromatographic run time. Preliminary results to obtain baseline resolution between monomer and aggregates were delineated previously [16]. This study should facilitate further investigation into purification performance of CHT chromatography as a tool to ensure elimination of aggregate in the purification of monoclonal antibody. 2. Experimental 2.1. Chemicals Ceramic hydroxyapatite (CHT) Type I 20 m and 40 m were obtained from Bio-Rad Laboratories (Hercules, CA). All chemicals of the highest grade available were purchased from VWR (Brisbane, CA). The phosphate buffers were prepared with monobasic sodium phosphate in nanopure water and were titrated to target pH values with 1 M sodium hydroxide.
column dimensions were 0.7 cm × 2.6 cm. The column was dry packed using a density of 0.60 g/mL. Each column was wetted in ten column volumes of 20% ethanol before equilibration. All chromatography experiments were automated and executed using the Biologic DuoFlow System and software. To determine the elution profile of immunoglobulin, chromatography was carried out with a sodium chloride gradient at target pH. As an example, the column was equilibrated in 0.005 M sodium phosphate pH 6.5 (buffer A). After IgG injection, usually 50 L, the column was washed with ten column volumes of equilibration buffer. The IgG was eluted using a gradient in sodium chloride concentration to 1.5 M (buffer B). This was achieved by programming the chromatography system to go from 0 to 100% B in 40 column volumes. The column was cleaned with 0.5 M sodium phosphate pH 6.5 prior to equilibration before each run. For imidazole containing buffers, imidazole was added to both buffers A and B and pH titrated with 6 M hydrochloric acid. For screening purposes, 0.3 M imidazole was selected since it is known that this concentration is more than sufficient to quench the histidine interaction between a protein and immobilized metal chelate complex [17,18]. For the elution profile of Thr 51, the column was initially equilibrated in 0.005 M sodium phosphate pH 5.9 (buffer A). After injection of the protein, the column was washed with ten column volumes of equilibration buffer. Thr 51 was eluted in 40 column volumes using a gradient in sodium phosphate concentration to 0.5 M pH 5.9 (buffer B). The column was stripped of any residual protein with ten column volumes of buffer B. From the elution profiles, % B was determined using the post run trace visibility provided by the DuoFlow software. The experiments were repeated using pH 6.5, 7.0 and 8.1. 2.4. Resolution factor (R)
2.2. Proteins Protein A eluate: clarified tissue culture fluid obtained from Avid BioSciences (Tustin, CA) was applied to a Millipore ProSep-A column. IgG1 was obtained by elution with 0.1 M glycine, 0.05 M sodium chloride, pH 3.8. The eluate was determined to contain a monomer/aggregate ratio of about 60:40 by size exclusion chromatography (HPSEC) on a Bio-Sil SEC 400-5 column (Bio-Rad Laboratories, Hercules, CA). Polyclonal human IgG was purchased from Sigma–Aldrich (St. Louis, MO). Purified monoclonal human IgG1 or IgG4 was obtained from clarified tissue culture fluid using protein A chromatography. The eluates contained >95% monomer by HPSEC. 51 Thr, a glutathione-S-transferase (GST) fusion protein with or without a C-terminal poly(6)histidine tag, was produced and purified at Bio-Rad Laboratories, Hercules, CA. The protein was determined to have a molecular weight of 51,000 Da. 2.3. Chromatography The experiments were conducted on a Bio Scale MT2 column obtained from Bio-Rad Laboratories (Hercules, CA). The
The resolution factor reflects the quality of separation between the monomer (A) and the aggregates (B). It is determined according to the equation, Rs = 2[tB − tA ]/(WA + WB ), where t and W correspond to the retention time and peak width at baseline, respectively. A resolution value of 1.5 implies a complete separation of the two adjacent peaks. 2.5. Peak of monomer It was located by determination of % B in which the monomer’s eluted peak maximum appeared. 2.6. Frontal dynamic binding capacity (DBC) Frontal loading studies to 10% breakthrough for CHT was performed at 600 cm/hr. After equilibration, each column was set off-line at the beginning of the run to obtain the absorbance at 100% breakthrough. Absorbance values were determined at 280 nm for a polyclonal antibody solution with an absorbance value of 1.2. The antibody solution was loaded onto the column through the injector valve after attaining 100% breakthrough. DBC at 10% breakthrough was calculated by mul-
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
15
Fig. 1. SFD of screening experiments.
tiplying the antibody concentration in the load by the frontal volume.
bodies with diverse isoelectric points in a polyclonal mixture could contribute to the observation.
2.7. SFD
3.2. Effect of phosphate on DBC
SFD was employed to help predict what results are expected when process parameters are changed. The screening experiment was designed with two levels of pH: 6.5 and 7.5; two levels of phosphate concentrations: 0.005 M and 0. 02 M; two levels of imidazole concentrations: 0 M and 0.3 M. The lower and upper levels for pH were chosen to reflect normal operating levels of CHT. The experiments were arranged in 23 statistical factorial designs with a total of eight runs. And in addition, three runs using the center point conditions were also carried out. These runs are important to establish curvature trends and to estimate errors in measurement. Conditions for the eleven experiments are shown in Fig. 1. The effects of pH (factor 1), phosphate concentrations (factor 2) and imidazole concentrations (factor 3) on resolution and peak elution were explored using a DOE software package (Umetrics MODDE 7, Kinnelon, NJ)
Elution of IgG from CHT requires phosphate, which out competes protein carboxyl clusters for CHT calcium and interrupts phosphoryl cation exchange. This effect is analogous to that of other Hofmeister ions (e.g. chloride, acetate) which can disrupt the interaction with the P sites. Accordingly, presence of phosphate in the feed material should depress the binding of polyclonal antibody. In the experiment, breakthrough curves were generated by monitoring absorbance at 280 nm over phosphate concentrations of 5, 10, 15 and 20 mM. The buffers were maintained at pH 7.0 DBC was shown to be inversely proportional to phosphate concentrations. This finding is consistent with the observed phosphate-dependent elution effect from hydroxyapatite media over a wide range of proteins [1–4]. While it is important to keep the phosphate concentration substantially low for maximum binding of IgG, it must be noted that a minimum level of phosphate is required to reduce degradation (data not shown). In the present study, 5 mM appeared to be sufficient to provide good DBC and resin stability.
3. Results and discussion 3.1. Effect of pH on DBC of polyclonal antibody Using a purified polyclonal antibody solution, breakthrough curves were generated by monitoring absorbance at 280 nm over a pH range of 5.8 to 7.7. The range extends beyond the low end of the operating pH of 6.5 since the crystalline structure of HAP degrades rapidly as the pH approaches 5. As shown in Fig. 2, DBC appears to reach a maximum at 6.7 to 6.8. At pH below or above the optimum, binding capacity was reduced, apparently due to diminished interactions derived from phosphoryl cation exchange or charge replusion. Furthermore, a multitude of anti-
Fig. 2. DBC as a function of pH.
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P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
Table 1 Effect of imidazole and NaCl on migration of peak maximum % B at peak maximum
Shift index (%)
IgG1 Control 0.12 M NaCl 0.3 M imidazole
43 34 31
0 −21 −28
IgG4 Control 0.12 M NaCl 0.3 M imidazole
48 42 32
0 −13 −33
Shift index = (%B experiment − %B control)/(%B control).
3.3. Effect of imidazole on migration of antibody Antibody bound to a CHT column can be eluted with a sodium chloride gradient in the presence of low amounts of phosphate ions. Adding 0.3 M imidazole to both buffers A and B showed that the protein eluted earlier in the gradient. After replacing 0.3 M imidazole with 0.12 M sodium chloride to generate an identical conductivity in the buffers, the profile revealed that the retention mechanism was not similarly affected. The result is consistent with the fact that CHT chromatography is mechanistically different from regular ion exchange chromatography. Ion exchange interaction, which is electrostatic interaction, alone cannot fully explain the elution behavior of IgG on CHT column. The surface chemistry of CHT provides both cation exchange and calcium affinity properties which work in concert to bind and resolve proteins. In the present case where IgG binds significantly at pH close to neutrality suggest that cation exchange is not the dominant factor since rather low pH values and high ionic strengths are required to bind and elute IgG using the latter mechanism. Shift index, a mathematical relationship as shown in Table 1, was used to demonstrate the effect of each buffer set on retention of a purified monoclonal antibody (IgG1 or IgG4 ). Buffer containing imidazole generally elutes the protein earlier, suggesting the outcome of a composite effect of ionic strength and coordination interactions between IgG and calcium. 3.4. Effect of imidazole on DBC As discussed in the method for determining DBC, breakthrough curves were generated by monitoring absorbance at 280 nm over an imidazole concentration of 0–0.4 M. The breakthrough capacity of CHT for polyclonal antibody decreases as imidazole concentration increases (Fig. 3), presumably caused by disruption of electrostatic interactions. Effect reaches asymptotic level at 300 mM imidazole.
Fig. 3. Effect of imidazole on DBC of IgG.
revealed upon increases of imidazole concentration. It is apparent that DBC was not altered by adding the histidine moiety, imidazole, once the electrostatic interaction was removed. 3.6. Chromatography of 51 Thr across CHT After 51 Thr was adsorbed onto the CHT column, it was eluted with a phosphate gradient. pH values studied were 5.9, 6.5, 7.0 and 8.1, a range normally used in CHT chromatography. The elution profile of 51 Thr at each pH was virtually identical and similar to the result observed when the C-terminus of the protein was tagged with poly(6)histidine. Values of % B of each peak maximum were tabulated in Table 2. From these results, it appears that the addition of histidine tag did not produce any differential effect on the elution characteristics of the protein studied. The observation is independent of the four pH values studied. It suggested that C site interactions were dominant at high pH while P site interactions started to be contributory at low pH. It is tempting to speculate that the carboxyl groups on the protein overwhelm the effect derived from the imidazole moieties of poly(6)histidine tag. However, further study and analysis will be required to evaluate this. 3.7. Accuracy of measurement on resolution and peak maximum To test the precision of the factors used in SFD, we measured the resolution factor and location of peak maximum in three runs using the center point conditions. The CV was determined to be 8 and 0%, respectively. The three runs showed essentially superimposable profiles. Therefore, the data are reliable for comparing results among experiments required for the SFD analysis. Table 2 Effect of poly(6)histidine tag on elution of 51 Thr
3.5. Effect of NaCl and imidazole on DBC of polyclonal antibody
pH
Sodium chloride at 0.15 M dramatically decreases DBC from approx 30 mg/mL as shown previously to 2 mg/mL. The purpose of the high level of NaCl in the equilibration buffer was to quench any electrostatic interaction. No additional effect on DBC was
5.9 6.5 7.0 8.1
%B at peak maximum Minus histidine tag
Plus histidine tag
53 31 24 15
54 31 23 15
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
17
Table 3 SFD Results
pH 6.5 vs. 7.5 Phosphate 0.005 M vs. 0.02 M Imidazole 0 M vs. 0.3 M
Resolution
Location of peak maximum
Improved at pH 6.5 Improved at 5 mM phosphate No improvement in majority of the cases containing 0.3 M imidazole
Higher eluent concentration at pH 6.5 Higher eluent concentration at 5 mM phosphate Higher eluent concentration without imidazole
Fig. 4. Contour plot on interaction of pH and phosphate on resolution (values in white boxes).
3.8. Effect of phosphate, pH and imidazole concentrations on resolution The objective of our screening experiment was to determine which process factors, out of the candidates, have an important effect on purification performance when IgG1 with a monomer/aggregate ratio of 60:40 was chromatographed across CHT. As determined by Umetrics MODDE 7, the factors which play a major role in the resolution are pH and phosphate. Imidazole concentration plays a minor role and can be eliminated as not a statistically significant factor in the reaction. Both pH and phosphate have an inversely proportional influence upon the reaction, that is, to increase resolution, one must decrease both the pH and phosphate levels. This trend is shown in the contour plot (Fig. 4). Furthermore, there appears to be interaction between the pH and phosphate factors. This interaction is in the positive direction, meaning a proportional response. Since pI of the antibody is alkaline and the best pH for resolution in this study was 6.5, the finding was consistent with selection of a low pH value so that sufficiently large net charge differences were created between monomeric IgG and aggregates. 4. Conclusion Using both polyclonal and monoclonal antibodies, this study described the preliminary development work carried out to establish the effects of three factors: pH, phosphate and imidazole on dynamic binding capacity and migration of peak maximum. The factors selected were considered significant for
understanding phosphoryl cation exchange, carboxyl chelation and calcium–histidine interaction. The results were used as the basis for evaluation of binding mechanism of IgG. Statistical factorial design of experiments was successfully applied to determine the sensitivity of feed variables on IgG migration and the resolution of monomer and aggregates from a protein A eluate. This study illustrates how a small number of experiments can be used to determine operating condition that optimizes resolution. Based on the statistical analysis, the significant factors for resolution are pH and phosphate. Imidazole enhances the eluent concentration which resulted in early elution of the peak maximum. This enhancement was also observed in the elution of the aggregates, resulting in insignificant effect of imidazole on resolution of monomer and aggregates. A summary of the observations is shown in Table 3. By capitalizing on how pH and phosphate affect the interaction of IgG with CHT, it should be possible to develop a generic protocol that can be used to purify monoclonal antibody. To investigate the calcium–histidine interaction in CHT, we studied the chromatography pattern of a fusion protein with or without a poly(6)histidine tag. Migration of the peak maximum of the untagged protein was indistinguishable from that of the tagged protein. This observation was consistently observed at a pH range of 5.9 to 8.1, suggesting that the effect of calcium– histidine interaction is insignificant, at least in the model protein studied. Acknowledgements The authors would like to thank Joshua Kellogg for his assistance with analysis and presentation of DOE data. The referee’s critical comments and valuable suggestions are greatly appreciated. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
M. Gorbunoff, Anal. Biochem. 136 (1984) 425. M. Gorbunoff, Anal. Biochem. 136 (1984) 433. M. Gorbunoff, S. Timasheff, Anal. Biochem. 136 (1984) 440. P. Gagnon, Purification Tools for Monoclonal Antibodies, Validated Biosystems, Inc., Tucson, AZ, 1996, p. 87. T. Kawasaki, J. Chromatogr. 544 (1991) 147. P. Chakrabarti, Protein Eng. 4 (1990) 57. C. Generus, D. Dehareng, B. Devreese, J. Van Beeumen, J. Frere, B. Joris, Biochem. J. 377 (2004) 111. M.F. Scully, V.V. Kakkar, Biochim. Biophys. Acta 700 (1982) 130. T.W. Hutchens, T.T. Yip, J. Chromatogr. 500 (1990) 531. R.V. Hogg, J. Ledolter, Engineering Statistics, third ed., MacMillan, New York, 1987. P.D. Haaland, Experimental Design in Biotechnology, Marcel Dekker, New York, 1989.
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[12] T.G. Cantu, E.W. Hoehn-Saric, K.M. Burgess, L. Racusen, P.J. Scheel, Am. J. Kidney Dis. 25 (1995) 228. [13] C. Pendley, A. Scharntz, C. Wagner, Cur. Op. Mol. Therapeutic. 5 (2003) 172. [14] E. Luellau, U. Stockar, S. Vogt, R. Freitag, J. Chromatogr. A 796 (1998) 165.
[15] T. Andersson, L. Hagel, Anal. Biochem. 141 (1984) 461. [16] P. Gagnon, P. Ng, J. Zhen, C. Aberin, J. He, H. Mekosh, L. Cummings, S. Zaidi, R. Richieri, Bioprocess Int. 4 (2006) 50. [17] E. Sulkowski, Trends Biotechnol. 3 (1985) 1. [18] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Nature 258 (1975) 598.
Mechanistic model for adsorption of immunoglobulin on hydroxyapatite Paul K. Ng a,∗ , Jie He a , Pete Gagnon b a
Process Applications, Process Chromatography Division, Life Sciences Group, Bio-Rad Laboratories, Hercules, CA 94547, USA b Validated Biosystems, San Clemente, CA 92672, USA Available online 11 December 2006
Abstract A model is developed for correlating the adsorption of protein to hydroxyapatite in terms of pH, phosphate and imidazole. It provides a basis for manipulating the chromatography conditions so as to obtain the best possible fractionation. The adsorption mechanism is apparently a function of two calcium affinity interactions and cation exchange. We used immunoglobulins and a fusion protein with or without a poly(6)histidine tag for our investigation. The results are presented and discussed in the present study. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxyapatite; IgG; Adsorption; Chromatography; Phosphate; pH; Resolution
1. Introduction Hydroxyapatite is a well-known multimodal chromatography support for purification of proteins and other biomolecules. While its multimodality and crystal structure are frequently credited for its unique selectivity, it also complicates screening and often leaves process developers at a loss with respect to optimizing a separation. Previous work with proteins such as IgG discussed retention on hydroxyapatite by a combination of two mechanisms: phosphoryl cation exchange which involves the interaction of positively charged amino groups on proteins with the P sites on the matrix; and calcium metal affinity which involves the protein carboxyl groups and the C sites on the matrix [1–4]. Using crystallography, Kawasaki [5] determined that the P sites and C cites are constructed from a single or several phosphate ions and a single or several crystal calcium ions, respectively. A third mechanism that has not been explored previously is metal–histidine interaction. Calcium ions can act as electrophiles, seeking to participate in electron pairs with other atoms so that a bond or charge–charge interaction can be formed. Such an interaction has been demonstrated between metal ions and the imidazole groups of histidyl side chains in a variety of proteins [6,7]. And in addition, affinity reten-
∗
Corresponding author. Tel.: +1 510 741 4839. E-mail address: paul [email protected] (P.K. Ng).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.071
tion of proteins by immobilized metal ions increased with the number of histidine residues [8,9]. Extension of these observations to IgG which carries multiple histidine residues on its complementarity-determining region (CDR) and the FC region suggests the possibility of histidine–calcium interaction as contributory to IgG retention. The present study attempts to determine the reactive constituents of the three mechanisms: phosphoryl cation exchange, carboxyl chelation and calcium–histidine interaction through the imidazole side chain of histidine residues. Results will be presented demonstrating the effect of pH, phosphate and imidazole on purification performance. Furthermore, this study applies statistical design to address the question, ‘what are the conditions for pH, phosphate and imidazole that optimize resolution?’. Statistical factorial design (SFD) provides the minimum number of experiments needed to evaluate the simultaneous effects of multiple variables and multi-variable interactions on predetermined processing objective [10,11]. Four stages are usually employed to apply SFD. First, ‘n’ variables that have an effect on process outcomes are selected. In our case these are pH, phosphate and imidazole. Second, anticipated ranges for each variable are identified for 2n experiments. Third, the process objectives are measured for each of the 2n experiments that span the variable ranges. Fourth, the effects of each variable and multi-variable interaction on predetermined process objectives are ascertained by commercially available Design of Experiments (DOE) software.
14
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
An important aspect of the present study is to facilitate investigation into purification performance of CHT chromatography as a tool to ensure elimination of aggregate in the purification of monoclonal antibody. Aggregation is one of the most important issues in the purification of monoclonal antibodies which are typically captured by protein A as the first chromatography step in a process train. Even with changes in the solvent conditions, such as adding surfactants, chaotropic agents and/or reducing agents or adjustment of wash conditions, presence of aggregates in the protein A eluate can still present significant technical challenge. These aggregates are known to be covalent or non-covalent interaction of denatured molecules. They can cause a loss of activity, reduced therapeutic value and untoward clinical effect [12,13]. It is therefore important to define process step to ensure its removal. Method such as size exclusion chromatography has been evaluated and was found to be laborious [14,15] because of low capacity and long chromatographic run time. Preliminary results to obtain baseline resolution between monomer and aggregates were delineated previously [16]. This study should facilitate further investigation into purification performance of CHT chromatography as a tool to ensure elimination of aggregate in the purification of monoclonal antibody. 2. Experimental 2.1. Chemicals Ceramic hydroxyapatite (CHT) Type I 20 m and 40 m were obtained from Bio-Rad Laboratories (Hercules, CA). All chemicals of the highest grade available were purchased from VWR (Brisbane, CA). The phosphate buffers were prepared with monobasic sodium phosphate in nanopure water and were titrated to target pH values with 1 M sodium hydroxide.
column dimensions were 0.7 cm × 2.6 cm. The column was dry packed using a density of 0.60 g/mL. Each column was wetted in ten column volumes of 20% ethanol before equilibration. All chromatography experiments were automated and executed using the Biologic DuoFlow System and software. To determine the elution profile of immunoglobulin, chromatography was carried out with a sodium chloride gradient at target pH. As an example, the column was equilibrated in 0.005 M sodium phosphate pH 6.5 (buffer A). After IgG injection, usually 50 L, the column was washed with ten column volumes of equilibration buffer. The IgG was eluted using a gradient in sodium chloride concentration to 1.5 M (buffer B). This was achieved by programming the chromatography system to go from 0 to 100% B in 40 column volumes. The column was cleaned with 0.5 M sodium phosphate pH 6.5 prior to equilibration before each run. For imidazole containing buffers, imidazole was added to both buffers A and B and pH titrated with 6 M hydrochloric acid. For screening purposes, 0.3 M imidazole was selected since it is known that this concentration is more than sufficient to quench the histidine interaction between a protein and immobilized metal chelate complex [17,18]. For the elution profile of Thr 51, the column was initially equilibrated in 0.005 M sodium phosphate pH 5.9 (buffer A). After injection of the protein, the column was washed with ten column volumes of equilibration buffer. Thr 51 was eluted in 40 column volumes using a gradient in sodium phosphate concentration to 0.5 M pH 5.9 (buffer B). The column was stripped of any residual protein with ten column volumes of buffer B. From the elution profiles, % B was determined using the post run trace visibility provided by the DuoFlow software. The experiments were repeated using pH 6.5, 7.0 and 8.1. 2.4. Resolution factor (R)
2.2. Proteins Protein A eluate: clarified tissue culture fluid obtained from Avid BioSciences (Tustin, CA) was applied to a Millipore ProSep-A column. IgG1 was obtained by elution with 0.1 M glycine, 0.05 M sodium chloride, pH 3.8. The eluate was determined to contain a monomer/aggregate ratio of about 60:40 by size exclusion chromatography (HPSEC) on a Bio-Sil SEC 400-5 column (Bio-Rad Laboratories, Hercules, CA). Polyclonal human IgG was purchased from Sigma–Aldrich (St. Louis, MO). Purified monoclonal human IgG1 or IgG4 was obtained from clarified tissue culture fluid using protein A chromatography. The eluates contained >95% monomer by HPSEC. 51 Thr, a glutathione-S-transferase (GST) fusion protein with or without a C-terminal poly(6)histidine tag, was produced and purified at Bio-Rad Laboratories, Hercules, CA. The protein was determined to have a molecular weight of 51,000 Da. 2.3. Chromatography The experiments were conducted on a Bio Scale MT2 column obtained from Bio-Rad Laboratories (Hercules, CA). The
The resolution factor reflects the quality of separation between the monomer (A) and the aggregates (B). It is determined according to the equation, Rs = 2[tB − tA ]/(WA + WB ), where t and W correspond to the retention time and peak width at baseline, respectively. A resolution value of 1.5 implies a complete separation of the two adjacent peaks. 2.5. Peak of monomer It was located by determination of % B in which the monomer’s eluted peak maximum appeared. 2.6. Frontal dynamic binding capacity (DBC) Frontal loading studies to 10% breakthrough for CHT was performed at 600 cm/hr. After equilibration, each column was set off-line at the beginning of the run to obtain the absorbance at 100% breakthrough. Absorbance values were determined at 280 nm for a polyclonal antibody solution with an absorbance value of 1.2. The antibody solution was loaded onto the column through the injector valve after attaining 100% breakthrough. DBC at 10% breakthrough was calculated by mul-
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
15
Fig. 1. SFD of screening experiments.
tiplying the antibody concentration in the load by the frontal volume.
bodies with diverse isoelectric points in a polyclonal mixture could contribute to the observation.
2.7. SFD
3.2. Effect of phosphate on DBC
SFD was employed to help predict what results are expected when process parameters are changed. The screening experiment was designed with two levels of pH: 6.5 and 7.5; two levels of phosphate concentrations: 0.005 M and 0. 02 M; two levels of imidazole concentrations: 0 M and 0.3 M. The lower and upper levels for pH were chosen to reflect normal operating levels of CHT. The experiments were arranged in 23 statistical factorial designs with a total of eight runs. And in addition, three runs using the center point conditions were also carried out. These runs are important to establish curvature trends and to estimate errors in measurement. Conditions for the eleven experiments are shown in Fig. 1. The effects of pH (factor 1), phosphate concentrations (factor 2) and imidazole concentrations (factor 3) on resolution and peak elution were explored using a DOE software package (Umetrics MODDE 7, Kinnelon, NJ)
Elution of IgG from CHT requires phosphate, which out competes protein carboxyl clusters for CHT calcium and interrupts phosphoryl cation exchange. This effect is analogous to that of other Hofmeister ions (e.g. chloride, acetate) which can disrupt the interaction with the P sites. Accordingly, presence of phosphate in the feed material should depress the binding of polyclonal antibody. In the experiment, breakthrough curves were generated by monitoring absorbance at 280 nm over phosphate concentrations of 5, 10, 15 and 20 mM. The buffers were maintained at pH 7.0 DBC was shown to be inversely proportional to phosphate concentrations. This finding is consistent with the observed phosphate-dependent elution effect from hydroxyapatite media over a wide range of proteins [1–4]. While it is important to keep the phosphate concentration substantially low for maximum binding of IgG, it must be noted that a minimum level of phosphate is required to reduce degradation (data not shown). In the present study, 5 mM appeared to be sufficient to provide good DBC and resin stability.
3. Results and discussion 3.1. Effect of pH on DBC of polyclonal antibody Using a purified polyclonal antibody solution, breakthrough curves were generated by monitoring absorbance at 280 nm over a pH range of 5.8 to 7.7. The range extends beyond the low end of the operating pH of 6.5 since the crystalline structure of HAP degrades rapidly as the pH approaches 5. As shown in Fig. 2, DBC appears to reach a maximum at 6.7 to 6.8. At pH below or above the optimum, binding capacity was reduced, apparently due to diminished interactions derived from phosphoryl cation exchange or charge replusion. Furthermore, a multitude of anti-
Fig. 2. DBC as a function of pH.
16
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
Table 1 Effect of imidazole and NaCl on migration of peak maximum % B at peak maximum
Shift index (%)
IgG1 Control 0.12 M NaCl 0.3 M imidazole
43 34 31
0 −21 −28
IgG4 Control 0.12 M NaCl 0.3 M imidazole
48 42 32
0 −13 −33
Shift index = (%B experiment − %B control)/(%B control).
3.3. Effect of imidazole on migration of antibody Antibody bound to a CHT column can be eluted with a sodium chloride gradient in the presence of low amounts of phosphate ions. Adding 0.3 M imidazole to both buffers A and B showed that the protein eluted earlier in the gradient. After replacing 0.3 M imidazole with 0.12 M sodium chloride to generate an identical conductivity in the buffers, the profile revealed that the retention mechanism was not similarly affected. The result is consistent with the fact that CHT chromatography is mechanistically different from regular ion exchange chromatography. Ion exchange interaction, which is electrostatic interaction, alone cannot fully explain the elution behavior of IgG on CHT column. The surface chemistry of CHT provides both cation exchange and calcium affinity properties which work in concert to bind and resolve proteins. In the present case where IgG binds significantly at pH close to neutrality suggest that cation exchange is not the dominant factor since rather low pH values and high ionic strengths are required to bind and elute IgG using the latter mechanism. Shift index, a mathematical relationship as shown in Table 1, was used to demonstrate the effect of each buffer set on retention of a purified monoclonal antibody (IgG1 or IgG4 ). Buffer containing imidazole generally elutes the protein earlier, suggesting the outcome of a composite effect of ionic strength and coordination interactions between IgG and calcium. 3.4. Effect of imidazole on DBC As discussed in the method for determining DBC, breakthrough curves were generated by monitoring absorbance at 280 nm over an imidazole concentration of 0–0.4 M. The breakthrough capacity of CHT for polyclonal antibody decreases as imidazole concentration increases (Fig. 3), presumably caused by disruption of electrostatic interactions. Effect reaches asymptotic level at 300 mM imidazole.
Fig. 3. Effect of imidazole on DBC of IgG.
revealed upon increases of imidazole concentration. It is apparent that DBC was not altered by adding the histidine moiety, imidazole, once the electrostatic interaction was removed. 3.6. Chromatography of 51 Thr across CHT After 51 Thr was adsorbed onto the CHT column, it was eluted with a phosphate gradient. pH values studied were 5.9, 6.5, 7.0 and 8.1, a range normally used in CHT chromatography. The elution profile of 51 Thr at each pH was virtually identical and similar to the result observed when the C-terminus of the protein was tagged with poly(6)histidine. Values of % B of each peak maximum were tabulated in Table 2. From these results, it appears that the addition of histidine tag did not produce any differential effect on the elution characteristics of the protein studied. The observation is independent of the four pH values studied. It suggested that C site interactions were dominant at high pH while P site interactions started to be contributory at low pH. It is tempting to speculate that the carboxyl groups on the protein overwhelm the effect derived from the imidazole moieties of poly(6)histidine tag. However, further study and analysis will be required to evaluate this. 3.7. Accuracy of measurement on resolution and peak maximum To test the precision of the factors used in SFD, we measured the resolution factor and location of peak maximum in three runs using the center point conditions. The CV was determined to be 8 and 0%, respectively. The three runs showed essentially superimposable profiles. Therefore, the data are reliable for comparing results among experiments required for the SFD analysis. Table 2 Effect of poly(6)histidine tag on elution of 51 Thr
3.5. Effect of NaCl and imidazole on DBC of polyclonal antibody
pH
Sodium chloride at 0.15 M dramatically decreases DBC from approx 30 mg/mL as shown previously to 2 mg/mL. The purpose of the high level of NaCl in the equilibration buffer was to quench any electrostatic interaction. No additional effect on DBC was
5.9 6.5 7.0 8.1
%B at peak maximum Minus histidine tag
Plus histidine tag
53 31 24 15
54 31 23 15
P.K. Ng et al. / J. Chromatogr. A 1142 (2007) 13–18
17
Table 3 SFD Results
pH 6.5 vs. 7.5 Phosphate 0.005 M vs. 0.02 M Imidazole 0 M vs. 0.3 M
Resolution
Location of peak maximum
Improved at pH 6.5 Improved at 5 mM phosphate No improvement in majority of the cases containing 0.3 M imidazole
Higher eluent concentration at pH 6.5 Higher eluent concentration at 5 mM phosphate Higher eluent concentration without imidazole
Fig. 4. Contour plot on interaction of pH and phosphate on resolution (values in white boxes).
3.8. Effect of phosphate, pH and imidazole concentrations on resolution The objective of our screening experiment was to determine which process factors, out of the candidates, have an important effect on purification performance when IgG1 with a monomer/aggregate ratio of 60:40 was chromatographed across CHT. As determined by Umetrics MODDE 7, the factors which play a major role in the resolution are pH and phosphate. Imidazole concentration plays a minor role and can be eliminated as not a statistically significant factor in the reaction. Both pH and phosphate have an inversely proportional influence upon the reaction, that is, to increase resolution, one must decrease both the pH and phosphate levels. This trend is shown in the contour plot (Fig. 4). Furthermore, there appears to be interaction between the pH and phosphate factors. This interaction is in the positive direction, meaning a proportional response. Since pI of the antibody is alkaline and the best pH for resolution in this study was 6.5, the finding was consistent with selection of a low pH value so that sufficiently large net charge differences were created between monomeric IgG and aggregates. 4. Conclusion Using both polyclonal and monoclonal antibodies, this study described the preliminary development work carried out to establish the effects of three factors: pH, phosphate and imidazole on dynamic binding capacity and migration of peak maximum. The factors selected were considered significant for
understanding phosphoryl cation exchange, carboxyl chelation and calcium–histidine interaction. The results were used as the basis for evaluation of binding mechanism of IgG. Statistical factorial design of experiments was successfully applied to determine the sensitivity of feed variables on IgG migration and the resolution of monomer and aggregates from a protein A eluate. This study illustrates how a small number of experiments can be used to determine operating condition that optimizes resolution. Based on the statistical analysis, the significant factors for resolution are pH and phosphate. Imidazole enhances the eluent concentration which resulted in early elution of the peak maximum. This enhancement was also observed in the elution of the aggregates, resulting in insignificant effect of imidazole on resolution of monomer and aggregates. A summary of the observations is shown in Table 3. By capitalizing on how pH and phosphate affect the interaction of IgG with CHT, it should be possible to develop a generic protocol that can be used to purify monoclonal antibody. To investigate the calcium–histidine interaction in CHT, we studied the chromatography pattern of a fusion protein with or without a poly(6)histidine tag. Migration of the peak maximum of the untagged protein was indistinguishable from that of the tagged protein. This observation was consistently observed at a pH range of 5.9 to 8.1, suggesting that the effect of calcium– histidine interaction is insignificant, at least in the model protein studied. Acknowledgements The authors would like to thank Joshua Kellogg for his assistance with analysis and presentation of DOE data. The referee’s critical comments and valuable suggestions are greatly appreciated. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
M. Gorbunoff, Anal. Biochem. 136 (1984) 425. M. Gorbunoff, Anal. Biochem. 136 (1984) 433. M. Gorbunoff, S. Timasheff, Anal. Biochem. 136 (1984) 440. P. Gagnon, Purification Tools for Monoclonal Antibodies, Validated Biosystems, Inc., Tucson, AZ, 1996, p. 87. T. Kawasaki, J. Chromatogr. 544 (1991) 147. P. Chakrabarti, Protein Eng. 4 (1990) 57. C. Generus, D. Dehareng, B. Devreese, J. Van Beeumen, J. Frere, B. Joris, Biochem. J. 377 (2004) 111. M.F. Scully, V.V. Kakkar, Biochim. Biophys. Acta 700 (1982) 130. T.W. Hutchens, T.T. Yip, J. Chromatogr. 500 (1990) 531. R.V. Hogg, J. Ledolter, Engineering Statistics, third ed., MacMillan, New York, 1987. P.D. Haaland, Experimental Design in Biotechnology, Marcel Dekker, New York, 1989.
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