Kinetic Studies on the Immobilization of Antibodies to High

The overall immobilization rate was essentially independent of the density of the support's activated sites (when present at a coverage of 0.1-0.4 μm...
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Bioconjugate Chem. 1998, 9, 459−465

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Kinetic Studies on the Immobilization of Antibodies to High-Performance Liquid Chromatographic Supports Matthew R. Oates, William Clarke, Elizabeth M. Marsh, and David S. Hage* Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304. Received August 17, 1997; Revised Manuscript Received January 14, 1998

Several factors can potentially affect the rate of immobilization of proteins onto solid supports, such as those used in affinity-based high-performance liquid chromatography. This study examined several of these factors and their influence on the coupling of periodate-treated rabbit immunoglobulin G antibodies to dihydrazide-activated silica. Items considered included the number of potential coupling sites on the antibodies, the density of activated sites on the support, the relative amount of antibody combined with the support, and the density of the overall reaction slurry. In each case, the rate of change in the solution-phase antibody concentration gave biphasic behavior which could be described by two competing pseudo-first-order reactions. The overall immobilization rate was essentially independent of the density of the support’s activated sites (when present at a coverage of 0.1-0.4 µmol/m2) but was strongly influenced by the number of available coupling groups on the antibodies. Increasing the slurry density had no appreciable effect on the immobilization rate, and the reaction rate showed only a small change when using different types of reagents for support activation (e.g., adipic vs oxalic dihydrazide). These results are consistent with a mechanism in which the rate-limiting step during immobilization is the covalent attachment of antibodies to the support and not mass transfer of antibodies to the support’s surface.

INTRODUCTION

Affinity chromatographic supports, such as those based on immobilized antibodies, are playing an ever-increasing role in the development of new separation and analysis methods for compounds of biological, industrial, or environmental interest (1-5). In recent years, the development of high-efficiency supports with good biocompatibility (e.g., diol-bonded silica or perfusion-type media) has promoted the use of various affinity ligands in highperformance liquid chromatography (HPLC)1 (1-7). This, in turn, has made affinity supports even more attractive for advanced analytical methods such as immunoanalytical assays, on-line monitoring methods for biotechnology or environmental testing, and multidimensional schemes in which affinity extraction is coupled with methods like reversed-phase or size-exclusion chromatography (3-5). There are a variety of procedures available for attaching proteins and other ligands to HPLC supports for use in affinity columns (1-3, 7). However, there is little information available in the literature regarding the immobilization kinetics of these methods. Such data would be valuable in the design and optimization of new HPLC affinity media, particularly as the demand for more efficient supports and more rapid method development increases. There have been numerous studies in the past that have examined the kinetic processes behind the physical adsorption of proteins to various solid matrixes (8-21), including chromatographic materials such as silica (e.g., see refs 17-21). The general kinetic model used in these studies involves two * To whom correspondence should be addressed. 1 Abbreviations: BCA, bicinchoninic acid; FIA, flow injection analysis; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; LyCH, lucifer yellow CH; TNBS, 2,4,6trinitrobenzenesulfonic acid.

initial steps: (1) mass transfer of protein to the liquidsolid interface and (2) adsorption of the protein to unoccupied regions on the support’s surface; a similar model would be expected to hold for covalent protein immobilization but with the adsorption step now being replaced with one involving the coupling of protein to the surface of the support. In many cases, the physical adsorption of proteins to silica and other supports has been reported to occur at a rate that is limited mainly by protein mass transfer to the surface (i.e., a diffusionlimited system) (8, 14-17), but there have also been cases in which adsorption has been noted to be the rate-limiting step (10, 12). The relative importance of these steps in determining the overall rate for the covalent immobilization of antibodies will be one item investigated in this work. This study will examine the rate of antibody immobilization onto HPLC-grade media by using periodatetreated rabbit immunoglobulin G as the model ligand and dihydrazide-activated silica as the model support. This particular system is of practical importance since antibodies are frequently used as ligands in affinity-based HPLC (3-5), and dihydrazide-activated supports are commonly used for their immobilization (7, 22-25). Furthermore, previous work has shown that several important experimental variables, such as the number of potential coupling sites available on each antibody and the surface coverage of active sites on the support, can be controlled in this system through the proper selection of conditions for the periodate oxidation of antibodies (24, 25) or for the preparation of dihydrazide-activated silica (23). Along with these variables, other factors that will be considered in this study with regard to immobilization rates will include the effect of varying the relative amount of antibody in contact with the support and the density of the overall reaction slurry. Such work should

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provide information on the overall importance of these factors in determining the net rate of antibody immobilization. The same data should also provide clues regarding the roles played by mass transfer versus covalent attachment in the immobilization kinetics of antibodies and other proteins. EXPERIMENTAL PROCEDURES

Reagents. Oxalic dihydrazide, adipic dihydrazide, and Lucifer yellow CH (LyCH) were from Aldrich (Milwaukee, WI). HPLC-grade Nucleosil Si-1000 silica (7 µm particle diameter, 1000 Å pore size, 25 m2/g surface area) was obtained from Alltech (Deerfield, IL). The rabbit immunoglobulin G (IgG), p-periodic acid reagent (i.e., periodic acid), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were from Sigma (St. Louis, MO). Ethylene glycol and Triton X-100 were obtained from Fisher Scientific (Pittsburgh, PA). Reagents for the bicinchoninic acid (BCA) protein assay were from Pierce (Rockford, IL). All other chemicals were reagent-grade or better. All aqueous solutions were prepared using deionized water from a Nanopure water system (Barnstead, Dubuque, IA). Apparatus. Samples for the manual BCA protein assay and TNBS assay were analyzed using disposable polystyrene cuvettes (Fisher Scientific) and a Shimadzu UV160U absorbance spectrophotometer (Kyoto, Japan). The degree of antibody oxidation was determined by labeling the oxidized antibodies with LyCH (26) and analyzing the degree of LyCH conjugation by flow injection analysis (FIA), as described previously (27). The application buffer for the FIA system was delivered by a Xper-Chrom model 400 pump from P. J. Cobert (St. Louis, MO); the BCA reagent for the same system was delivered by a model J120 cassette pump from Manostat (New York, NY). The LyCH label was detected at 428 nm on the FIA system by using a SM3100X absorbance detector from LDC/Milton Roy (Riviera Beach, FL), and the colored BCA product was detected at 562 nm using a V4 absorbance detector from ISCO (Lincoln, NE). The FIA samples were injected using a Rheodyne 7012 injection valve (Cotati, CA) equipped with a 20 µL sample loop. The BCA reagent was combined with samples in the FIA system by using a standard mixing tee (Upchurch, Oak Harbor, WA), followed by passage of the mixture through a serpentine type II reaction chamber made from 1.0 m × 0.5 mm inner diameter PTFE tubing (28). This reaction chamber was immersed in an adjustable water bath set at 80 °C. Methods. The diol-bonded silica and dihydrazideactivated supports were prepared from Nucleosil Si-1000, according to the scheme shown in Figure 1. The final diol coverage of the Nucleosil prior to activation by dihydrazide groups was 62 ( 4 µmol of diol per gram of silica, as determined in triplicate by an iodimetric capillary electrophoresis method (29). The oxalic and adipic dihydrazide-activated supports were both prepared from the same batch of diol-bonded silica. Various degrees of dihydrazide coverage were obtained on these supports by controlling the amount of diol oxidation that was used during support preparation, as described in ref 23. The final coverage of activated sites on each support was determined by reacting the final material with an excess of TNBS, followed by washing away of the excess reagent and measurement of the resulting colored product by suspending the treated silica in a saturated sucrose solution and determining the absorbance of the suspension at 425 nm (23).

Figure 1. Reactions for the synthesis of dihydrazide-activated silica (23). Table 1. Oxidation Conditions Used To Generate Rabbit IgG Antibodies with Various Levels of Potential Coupling Sites pH

[periodic acid] (mM)

oxidation time (min)

degree of oxidation (mol of LyCH/mol of IgG)a

5.0 7.0 5.0 4.0 3.0

0.05 10.0 10.0 10.0 10.0

10 10 30 60 60

0.6 ((0.1) 0.9 ((0.1) 2.0 ((0.1) 3.6 ((0.1) 4.6 ((0.1)

a The numbers in parentheses represent a range of (1 SD. The value of moles of LyCH per mole of IgG is a measure of the average number of reactive aldehyde groups per IgG molecule that are available for coupling to a hydrazide-containing reagent (24).

The number of potential coupling sites on the rabbit IgG antibodies was varied by altering the pH and time used to treat the antibodies with periodic acid (24). All antibody oxidation was carried out at 25 °C using a rabbit IgG concentration of 1 mg/mL in 0.02 M sodium acetate buffer containing 0.15 M sodium chloride; Table 1 lists the other conditions that were used for antibody oxidation. After each oxidation mixture had been reacted for the appropriate length of time, ethylene glycol was added to quench the reaction. The ethylene glycol and remaining periodate were then removed by size-exclusion chromatography and dialysis, as described earlier (26); during this process, the antibodies were placed into a pH 6.0, 0.10 M phosphate buffer for the immobilization step. The final oxidized antibody preparations were either used immediately or stored for short periods of time at 4 °C prior to being used. Antibody immobilization was carried out by mixing each oxidized antibody preparation with a fixed amount of dihydrazide-activated silica suspended in pH 6.0, 0.10 M phosphate buffer. This mixture was vortex-mixed, placed onto an inversion shaker, and allowed to react for various lengths of time at 4 °C. Aliquots were periodically removed from this mixture and centrifuged to remove the supernatant from the silica. The protein content in the supernatant was then determined in duplicate by a manual BCA assay (30), with rabbit IgG being used as the standard. Similar control studies were performed with nonoxidized antibodies in the presence of diol-bonded or dihydrazide-activated silica and with oxidized antibodies in the presence of diol-bonded silica.

Immobilization of Antibodies to Supports

Figure 2. Immobilization rate of rabbit IgG when the coverage of activated groups on the surface of adipic dihydrazide-activated silica was (4) 3.0, (]) 5.8, (+) 7.3, or (9) 10.5 µmol of reactive hydrazide groups per gram of silica. RESULTS AND DISCUSSION

Effect of Support Active Site Coverage on Antibody Immobilization Rate. The first series of studies examined the effect on the rate of antibody immobilization when the support’s surface coverage of active sites to which the antibody could be attached was changed. In our model system, the coverage of active hydrazide groups was varied by adjusting the amount of periodate used in converting diol-bonded silica into an aldehydeactivated form (i.e., see the second step in Figure 1) prior to reaction of this support with a dihydrazide reagent (23). The conditions used in this work involved converting 25-100% of the available diol groups into aldehydes for reaction with adipic dihydrazide; under these conditions, the final coverages obtained for active hydrazide groups ranged from 3 to 10.5 µmol/g of silica, or 0.120.42 µmol/m2, in agreement with previously reported results (23). Figure 2 shows the results obtained when a constant amount of oxidized rabbit IgG was reacted with fixed amounts of several silica samples that had various surface coverages of active hydrazide groups. The IgG was oxidized under conditions that produced two available aldehyde groups per antibody (see conditions in Table 1 for 2.0 mol of LyCH/mol of IgG); the total amount of added antibody was 2.2 µmol/L or 0.33 g/L (5 mg in 15 mL), and the amount of silica in the reaction slurry was 13.3 g/L (200 mg in 15 mL). Under these conditions, the active hydrazide groups were present in a 13-67-fold molar excess versus the antibodies, and the maximum degree of antibody immobilization that could be achieved was approximately 0.3 monolayer. It can be seen from Figure 2 that essentially the same rate of antibody immobilization was observed for each surface coverage of active hydrazide groups. The consistency of these results implies that, under these particular conditions, the immobilization rate of IgG was independent of the silica’s active group coverage. In other words, the immobilization rate appears to have had a zero-order dependence on the surface concentration of the support’s active sites. Similar control studies performed with nonoxidized rabbit IgG in the presence of either diolbonded silica or the dihydrazide-activated silica did not give rise to any observable immobilization; this indicated that the results in Figure 2 were due to specific covalent attachment rather than a process involving nonspecific physical adsorption of the antibodies to the silica surface (23).

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Figure 3. Immobilization rate observed for rabbit IgG for reaction mixtures containing (9) 2.2, (]) 1.1, or (4) 0.33 g/L IgG and 13.3 g/L adipic dihydrazide-activated silica. The total amount of IgG used in these experiments corresponded to maximum theoretical coverages on the support of approximately 2.0, 1.0, or 0.3 monolayers, respectively.

Although it may at first be surprising that the immobilization rate is essentially independent of the support’s active group coverage, this can be explained by considering the relative size of an antibody versus the average area occupied by an active site on the support. For example, rabbit IgG (MW of 150 kDa) has a Stoke’s diameter of approximately 100 Å (31), which gives this protein a cross-sectional area of about 7900 Å2 when it is described as a spherical molecule. In contrast to this, a support with an active site coverage of 0.1-0.4 µmol/ m2, as used in this work, would have an average area per active site ranging from 1600 to 400 Å2, respectively. This means that each rabbit IgG molecule will cover an area occupied by about 19-20 active sites (at 400 Å2/site) or 4-5 active sites (at 1600 Å2/site). The fact that there is a large molar excess of these active sites versus each antibody, and that these active groups are nonmobile, supports a model in which the coverage of these groups is not an important factor in limiting the number of effective collisions between the antibodies and the support during the immobilization process. At much lower surface coverages of the active sites, some dependence of the immobilization rate on active site coverage would be expected, but it is anticipated that the conditions required to achieve this are well below those typically used in affinity supports for HPLC (e.g., 0.02 µmol of active groups/m2 would be needed to obtain an average of only one active hydrazide site per 7900 Å2, the approximate cross-sectional area of rabbit IgG). Effect of Antibody Concentration on Immobilization Rate. A second factor considered in these immobilization studies was the role played by antibody concentration in determining the net rate of reaction. Figure 3 illustrates how antibody concentration affected the immobilization rate for rabbit IgG on adipic dihydrazide-activated silica. As in the previous section, the oxidized IgG contained about two available aldehyde groups per antibody and the amount of silica in the reaction slurry was 9-13 g/L; the degree of support activation was 3.0 µmol of hydrazide groups/g of silica for the 0.33 g/L IgG data and 10-11 µmol/g for the 1.12.2 g/L IgG results. The initial amount of rabbit IgG in solution for the experiments shown in Figure 3 ranged from 0.33 to 2.2 g/L; these levels correspond to maximum theoretical coverages on the support of 0.3-2 monolayers (i.e., antibody amounts that ranged from below that

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Figure 4. Relative change in solution-phase antibody concentration as a function of time for the immobilization of rabbit IgG to adipic dihydrazide-activated silica. The initial IgG concentration was 0.33 g/L; the level of silica was 13.3 g/L, and the degree of antibody oxidation was 2.0 reactive aldehydes/ mol of IgG. In this graph, [P]t represents the molar concentration of protein in the supernatant at time t, [P]o is the initial protein concentration in the supernatant, and [P]∞ is the amount of protein remaining in the supernatant after the immobilization reaction has reached completion. The inset shows an enlarged view of the data points obtained over the first 10 h of the reaction.

needed to saturate the support’s surface to amounts in excess of the surface area available for immobilization). The reaction profiles in Figure 3 (and also those shown earlier in Figure 2) are typical of those seen throughout our immobilization studies with rabbit IgG. Such profiles generally included a fast rate of antibody immobilization during the first day of the reaction, followed by a slower rate that proceeded over much longer times. This type of biphasic behavior has been noted over a shorter time scale for the physical adsorption of human serum albumin (9), lysozyme, β-lactoglobulin, and hemoglobin to silica (21) and for the adsorption of bovine serum albumin, IgG, fibrinogen, and polylysine to poly(vinyl chloride) or methylacrylic-methyacrylate copolymers (13). The immobilization reaction in this study was essentially complete after 12-14 days when working with antibody levels at or below a theoretical support coverage of 1 monolayer (see Figure 2 and data in Figure 3 at 0.33 and 1.1 g/L IgG). For higher amounts of antibody (see data in Figure 3 for 2.2 g/L IgG), the reaction was also complete after 12-14 days, with the excess antibodies remaining in solution as a nonimmobilized fraction. This time frame is several orders of magnitude longer than that observed in any previous studies for the physical adsorption of proteins (including antibodies and silica supports) under mass-transfer-limited conditions (8, 1421) and suggests a reaction mechanism in which the overall immobilization rate is controlled mainly by the rate of covalent attachment of antibodies to the support. A more detailed analysis of the antibody immobilization data by using log concentration versus time plots consistently gave a response with two linear regions (see Figure 4). This fits a model in which the net rate of antibody immobilization to dihydrazide-activated silica can be described by two competing pseudo-first-order processes; a similar model has previously been used by others to describe the physical adsorption of various proteins to silica (21). The data obtained by these plots for studies at relatively low antibody levels (e.g., the results in Figure 4 at 0.33 g/L rabbit IgG and 13.3 g/L

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silica) gave an apparent first-order rate constant for the fast immobilization reaction of approximately (1.9 ( 0.3) (one SD) × 10-5 s-1 (half-life, 10 h); the same data gave an apparent first-order rate constant for the second, slower immobilization reaction of (4 ( 1) (one SD) × 10-6 s-1 (half-life, 2 days). Similar rate constants were obtained at each of the other antibody levels that were tested in this study. As mentioned earlier, control experiments with diolbonded or dihydrazide-activated silica and nonoxidized rabbit IgG did not give rise to any observable antibody immobilization. This indicated that neither the fast nor slow immobilization reactions seen in Figure 4 were due to physical adsorption of the antibodies to the support. However, it was found that the combination of oxidized antibodies with diol-bonded silica did give rise to antibody attachment. In this case, a plot of the log of the antibody concentration versus time gave linear behavior (correlation coefficient ) 0.9813 for 18 data points obtained over 0-1.2 days), as seen earlier for the dihydrazide-activated supports, indicating that the reaction of oxidized antibodies with diol-bonded silica was also a pseudo-first-order process. Furthermore, the apparent rate constant for the oxidized antibody-diol-bonded silica reaction [(1.7 ( 0.1) × 10-5 s-1] was statistically identical to the value obtained in Figure 4 for the fast reaction of oxidized antibodies with dihydrazide-activated silica. This suggests that it is the remaining diol groups on the silica (and also perhaps primary alcohols that are generated in the third step of the reaction scheme in Figure 1) that are responsible for the fast immobilization process on both types of supports. Although further work is needed to conclusively identify the mechanism for this fast process, one strong possibility is that the aldehyde groups on the oxidized antibodies are forming acetal or hemiacetal linkages with secondary and primary alcohols on the surface of the silica. Regardless of whether this is the actual mechanism for the fast reaction, the results of this control study do indicate that the second, slower immobilization process seen for dihydrazide-activated silica is the one that actually represents the attachment of oxidized antibodies to active hydrazide groups on this support. Effect of Antibody Coupling Sites on Immobilization Rate. Besides antibody concentration, this report also considered how varying the number of potential coupling sites on an antibody affected its net rate of immobilization. This factor was controlled by varying the oxidation conditions used during the periodate treatment of antibodies prior to immobilization. As discussed in detail previously (24, 25), using different times, temperatures, pHs, or periodate concentrations for antibody oxidation can lead to the production of various amounts of active aldehyde groups on an antibody’s carbohydrate residues. This, in turn, leads to different numbers of sites at which hydrazide groups on the support can attach to the antibodies for immobilization. Figure 5 shows the results obtained for adipic and oxalic dihydrazide-activated silica supports when they were reacted with rabbit IgG containing an average of 0.6-4.6 available aldehyde groups per antibody molecule, as prepared according to the oxidation conditions shown in Table 1. The total amount of antibody used in each case was 0.33 g/L, and the silica content in the reaction slurry was 13.3 g/L. The coverages of active hydrazide groups on the adipic and oxalic dihydrazide-activated supports were both in the range of 5-6 µmol/g of silica (23). The general behavior of the plots in Figure 5 was the same as that noted in the previous studies (see

Immobilization of Antibodies to Supports

Figure 5. Immobilization rates observed for rabbit IgG containing various amounts of potential coupling sites in the presence of (a) adipic dihydrazide-activated silica or (b) oxalic dihydrazide-activated silica. In panels a and b, the number of potential coupling sites on the antibodies was (3) 0.6, (9) 0.9, (+) 2.0, (]) 3.6, or (×) 4.6 aldehydes per IgG.

Figures 2-4) in which a fast rate of antibody immobilization occurred during the first day of the reaction, followed by a slower immobilization rate over longer reaction times. Similar rates were observed for the oxalic and adipic dihydrazide supports when antibodies containing 0.6 available aldehyde per IgG were used, but a slightly faster immobilization rate was seen for the oxalic dihydrazide-activated support with antibodies that contained an average of 0.9-4.6 aldehydes per IgG. The exact reason for this apparent difference is still under investigation, but it is probably related to the different local environments that are known to be present for the immobilized oxalic and adipic dihydrazide groups. For example, it has been shown previously that these two reagents yield different fractions of active hydrazide residues as the density of aldehyde groups on silica surface is varied; this behavior is, in turn, related to the different chain lengths of these dihydrazide reagents and their different tendencies to undergo bifunctional attachment to aldehyde-activated silica (23). It can be seen in Figure 5 that, when the average number of aldehyde groups per antibody was less than one (e.g., 0.6 or 0.9 aldehyde/IgG), a significant fraction of antibodies remained in solution after 14 days of reaction. This occurred because not all of the antibodies possessed coupling sites (i.e., aldehyde groups) under such conditions. For example, the data obtained when working with antibodies that had an average of 0.6 aldehyde per IgG might represent a situation in which

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Figure 6. Plots of (a) the relative change in solution-phase antibody concentration as a function of time for the immobilization of rabbit IgG to adipic dihydrazide-activated silica for antibodies containing (3) 0.6, (9) 0.9, (+) 2.0, or (]) 3.6 available aldehyde groups per IgG and (b) the change in the apparent pseudo-first-order rate constant (kapp) for the slow immobilization reaction as a function of the degree of antibody oxidation. The data used in generating these plots were obtained from Figure 5a. All abbreviations used on the y-axis in panel a are the same as those defined in Figure 4.

just under half of the antibodies had no aldehyde groups and the remainder had only one such group per IgG (e.g., if all oxidation took place at only terminal sialic acid residues) (24). Because of this, it was only at higher degrees of oxidation (2.0-4.6 aldehydes per IgG) that essentially complete immobilization of all antibodies was observed. Examples of first-order kinetic plots that were obtained for the data in Figure 5 are shown in Figure 6. As discussed earlier, the general trend seen was one in which the reaction obeyed pseudo-first-order kinetics with respect to antibody concentration, with two competing reactions (one fast and one slow) being present. However, one interesting feature now observed was the fact that the reaction rates showed a strong dependence on the number of aldehyde groups available on the antibodies for coupling to the dihydrazide-activated support. This was most clearly seen for the slower immobilization process, in which the apparent pseudo-firstorder rate constant increased with the average number of aldehyde groups per IgG (see Figure 6b). Such behavior is an indication of the importance of an antibody having the correct orientation in obtaining an effective collision with the support for immobilization. In addition, this result further supports a mechanism in which the net rate of immobilization is controlled by the rate of covalent coupling between the antibody and the support.

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Figure 7. Change in the immobilization rate for rabbit IgG when using silica amounts in the reaction slurry of (+) 20, (]) 13, (×) 6.7, or (4) 3.3 g/L.

Effect of Slurry Density on Antibody Immobilization Rate. A final study was performed to determine the effect of varying the slurry density of the reaction mixture on the observed rate of antibody immobilization. The results are shown in Figure 7. These graphs were generated using antibodies containing an average of 2 aldehyde groups per IgG. The support was adipic dihydrazide-activated silica that contained 10-11 µmol of hydrazide groups per gram of silica. The slurry density was varied by adjusting the volume of buffer present in the reaction mixture while keeping a constant mass of added antibodies and silica (i.e., a ratio of 1 mg of rabbit IgG per 40 mg of silica or a maximum theoretical coverage of 0.3 monolayer for IgG on the silica’s surface). The data in Figure 7 show that only random changes occurred in the immobilization rate when support concentrations of 3.3-20 g/L and IgG concentrations of 0.080.5 g/L were used. Kinetic plots made with these data gave the same type of biphasic behavior seen in Figures 4 and 6a; the pseudo-first order rate constants obtained also agreed with those given in the previous sections. The consistency of the results in Figure 7 indicates that the slurry density was not an important factor in controlling the rate of antibody immobilization. This is again consistent with a reaction mechanism in which the net of rate of immobilization is controlled mainly by the rate of antibody attachment rather than by antibody mass transfer to the surface of the support. CONCLUSIONS

This work considered several factors that can potentially affect the immobilization rate of antibodies onto HPLC supports, using rabbit IgG and dihydrazideactivated silica as a model system. The overall immobilization rate was essentially independent of the density of the support’s activated sites (when present at a coverage of 0.1-0.4 µmol/m2) but was strongly influenced by the number of available coupling groups on the antibodies. Increasing the slurry density of the support and antibodies in the reaction mixture had little or no effect on the immobilization rate, and the reaction rate showed only a small change when different types of reagents were used for support activation (e.g., adipic vs oxalic dihydrazide). In each case studied, the rate of change in the solutionphase antibody concentration gave biphasic behavior which could be described by two competing pseudo-firstorder reactions. One of these reactions was relatively fast (half-life, 10 h), while the second proceeded at a much

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slower rate (half-life, 2 days). Through control experiments, it was found that both processes required the presence of antibody oxidation, indicating that these reactions were due to covalent linkages rather than due to simple physical adsorption. It was further determined that the fast and slow reactions were most likely the result of the coupling of oxidized antibodies with (1) secondary or primary alcohols on the support and (2) active hydrazide residues on the support, respectively. The results of this study provide some interesting insights into the kinetics of antibody immobilization and the factors which are most important in influencing the rates of such reactions. For example, the results of this work consistently indicated that antibody attachment (vs mass transfer) was the rate-limiting step in controlling the overall immobilization process; this behavior is much different from that generally seen for the physical adsorption of proteins to surfaces, a reaction which often tends to be mass-transfer-limited (8, 14-17). It is expected that many of the trends reported in this work apply not only to antibodies but also to other proteins during their immobilization to small diameter supports. The data provided in this study should also be useful in providing some initial guidelines regarding the selection of conditions for the preparation of affinity-based HPLC media. ACKNOWLEDGMENT

This work was supported in part by the National Institutes of Health through Grant RO1 GM44931. M.R.O. was supported in part by a fellowship from the University of Nebraska Center for Biotechnology. LITERATURE CITED (1) Larsson, P.-O. (1984) High-performance liquid affinity chromatography. Methods Enzymol. 104, 212-223. (2) Walters, R. R. (1985) Affinity chromatography. Anal. Chem. 57, 1099A-1114A. (3) Phillips, T. M. (1985) High performance immunoaffinity chromatography. LC Mag. 3, 962-972. (4) Phillips, T. M. (1989) High-performance immunoaffinity chromatography. Adv. Chromatogr. 29, 133-173. (5) De Frutos, M., and Regnier, F. E. (1993) Tandem chromatographic-immunological analyses. Anal. Chem. 65, 17A-25A. (6) Afeyan, N. B., Gordon, N. F., Mazsaroff, I., Varady, L., Fulton, S. P., Yang, Y. B., and Regnier, F. E. (1990) Flowthrough particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography. J. Chromatogr. 519, 1-29. (7) Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992) Immobilized affinity ligand techniques, Academic Press, New York. (8) Lok, B. K., Cheng, Y. L., and Robertson, C. R. (1982) Protein adsorption on crosslinked polydimethylsiloxane using total internal reflection fluorescence. J. Colloid Interface Sci. 91, 104-116. (9) Van Dulm, P., and Norde, W. (1983) The adsorption of human plasma albumin on solid surfaces, with special attention to the kinetic aspects. J. Colloid Interface Sci. 91, 248-255. (10) Cheng, Y. L., Darst, S. A., and Robertson, C. R. (1986) Bovine serum albumin adsorption and desorption rates on solid surfaces with varying surface properties. J. Colloid Interface Sci. 118, 212-223. (11) Hlady, V., Reinecke, D. R., and Andrade, J. D. (1986) Fluorescence of adsorbed protein layers. I. Quantitation of total internal reflection fluorescence. J. Colloid Interface Sci. 111, 555-569. (12) Young, B. R., Pitt, W. G., and Cooper, S. L. (1988) Protein adsorption on polymeric biomaterials. II. Adsorption kinetics. J. Colloid Interface Sci. 125, 246-260.

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