Small-Molecule-Based Affinity Chromatography Method for Antibody

Aug 28, 2012 - The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and ...
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Small-Molecule-Based Affinity Chromatography Method for Antibody Purification via Nucleotide Binding Site Targeting Nathan J. Alves,† Samuel D. Stimple,† Michael W. Handlogten,† Jonathan D. Ashley,† Tanyel Kiziltepe,†,‡ and Basar Bilgicer*,†,‡,§ †

Department of Chemical and Biomolecular Engineering, ‡Advanced Diagnostics and Therapeutics, and §Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States S Supporting Information *

ABSTRACT: The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a Kd ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBSIBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBSIBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBSIBA column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBSIBA column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBSIBA platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.

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denatured antibody, or high-molecular-weight antibody aggregates that remain in the purified antibody product, which cause decreased antibody activity.3−11 Finally, the standard nonoriented methods for immobilization of protein A/G to solid supports can result in significant loss of binding activity (in some cases as high as ∼96% according to reports) due to steric constraints, yielding reduced column capacity.12 Alternative approaches have been developed in the past decade to address some of the issues associated with protein A/ G columns including engineering protein A/G variants with increased stability13−16 and using alternative protein ligands such as camelid antibodies.17,18 Both of these approaches, however, are more expensive than the protein A/G resins and are not yet established in commercial scale production. Another alternative approach has been to develop small-molecule capture ligands, which have several advantages over protein ligands. Small molecules deliver elevated resistance to physical and chemical degradation, thereby prolonging column lifetime, and eliminate the leaching of column components into the purified antibody product. Immobilization of small molecules to the solid support is also site-specific, thereby minimizing the loss of activity and decreased column capacity associated with nonspecific protein immobilization. These significant advantages, combined with a much-reduced cost, have motivated the

ue to their extraordinary specificity and affinity for antigens, antibodies are the primary choice for use in a vast array of applications including detection, diagnosis, and clinical therapeutics. Particularly, in the last 15 years, monoclonal antibodies have revolutionized the treatment of many diseases including cancers, autoimmune diseases, and infectious diseases. Despite the clinical benefits and improved patient outcomes associated with antibody-based therapies, the high cost, which may reach several thousands of dollars per dose, makes the treatments unaffordable to many patients. Therefore, novel purification methods that will lower antibody production costs are urgently needed. A major contributor to the antibody production costs is the use of protein A or G affinity columns in the downstream processing, which have become the industry standard for antibody purification processes.1,2 In this method, immobilized protein A/G binds to the antibody Fc domains to remove contaminants such as proteins, DNA, endotoxins, and cell culture media additives. Although this technique is reported to yield >95% antibody purity, there are several problems associated with its use. One of the major problems is the limited stability of protein A/G under the acidic pH conditions that are necessary to elute the captured antibody and the alkaline conditions used for in situ resin sterilization. Repeated use of these harsh conditions results in (i) loss of protein A/G tertiary structure, which limits the column lifetime, (ii) leaching of protein A/G fragments, which causes contamination of the purified antibody, and most importantly (iii) misfolded, © 2012 American Chemical Society

Received: April 9, 2012 Accepted: August 28, 2012 Published: August 28, 2012 7721

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Figure 1. (A) Location of the nucleotide binding site (NBS) is shown on the crystal structure of the antibody Fab variable domain. (B) Cartoon representation of antibody capture by IBA-conjugated ToyoPearl resin.

purchased from Cell-Gro (Manassas, VA), and fetal bovine serum (FBS) was from Hyclone (Thermo Scientific, Rockford, IL). Rituximab and trastuzumab were gifts from Dr. Navari at the Indiana University School of Medicine in South Bend, IN. Resin Synthesis and Preparation. The IBA-functionalized ToyoPearl AF-Amino-650 M (NBSIBA) resin, nonfunctionalized acetylated control resin, and unmodified control resin were prepared as described by the manufacturerrecommended coupling procedure for carboxylic acid containing ligand immobilization using EDC/NHS. Briefly, the ligand solution was prepared in distilled water and then added to the 0.5 M NaCl pH 4.5 rinsed resin. EDC and NHS (30 mg/mL of gel) were then added to the mixture, and the reaction was carried out on a shaker for 24 h at 25 °C. Post coupling, the resin was rinsed with 1.0 M NaCl to remove any unreacted ligand. A quantitative ninhydrin test was used to quantify IBA loading on the resin based on the remaining number of unreacted amines. All resins were packed via gravity settling of a 50% v/v slurry in 2 M NaCl pH 7.0 elution buffer in a C-1035 (2 μm, double-fritted, 10 mm i.d. × 10 mm in length, 785 μL) cylindrical column (Idex HS, Oak Harbor, WA). The column was placed into a C-1000 cartridge holder and attached to a liquid chromatography (LC) system. A pressure drop of 3 bar across the column was applied to pack the resin by flowing elution buffer for 2 h. The column was then equilibrated by flowing equilibration buffer at 0.5 mL/min for 1 h. Buffers and Gradient Used for Affinity Chromatography Separations. An Agilent Technologies 1200 Series HPLC system was used in all chromatographic injections. The pressure drop across the column ranged between 1 and 3 bar over the course of the NaCl elution gradient at a constant flow rate of 0.5 mL/min. Unless otherwise noted (i) following injection of sample, the column was washed for 5 min with EQ buffer (equilibration buffer, 50 mM phosphate buffer at pH 7.0) to capture antibody and wash away contaminants, (ii) the antibody was then eluted using a 10 min linear gradient from 0 to 100% ELS buffer (elution buffer, 2 M NaCl in 50 mM phosphate buffer at pH 7.0), (iii) the column was cleaned with ELS buffer for 3 min and (iv) re-equilibrated for 11 min with EQ buffer. When indicated, the flow through (contaminants eluting between 0.5 and 4.0 min) and elution (purified antibody 12.0−15.5 min) fractions were collected for further analysis. Determination of Antibody Recovery and Purity by SEC. A Tosoh Biosciences G4000SWXL size exclusion column was used to assess purity of the antibody. The flow through and elution fractions collected from NBSIBA column separations were first concentrated 25-fold using 0.5 mL, 10 kDa cutoff,

development of several synthetic ligands for antibody purification, albeit with limited market success largely due to their lack of specificity.19−25 Therefore, development of a novel method for antibody purification with reduced cost, enhanced stability, and improved specificity is urgently needed. In this study, we report a novel affinity chromatography method for purification of antibodies. Our method utilizes the not-so-known unconventional nucleotide binding site (NBS) present on the antibody Fab arms as a target for capturing antibodies on an affinity column. In an earlier publication, we demonstrated that the NBS is a highly conserved binding pocket present on all antibody isotypes across various species and identified a small-molecule ligandindole-3-butryic acid (IBA)that selectively binds to the NBS (Figure 1A).26,27 In the herein described method, we utilize the NBS to selectively capture and purify antibodies by conjugating IBA to ToyoPearl resin to generate an NBS targeting affinity column (NBSIBA) (Figure 1B). Evaluation of the NBSIBA column using the chimeric antibody rituximab established that monoclonal antibodies were selectively captured and retained on the NBSIBA column. Antibody recovery was consistently >95%, with >98% purity in the presence of various contaminant sources such as bovine serum albumin (BSA), hybridoma media, conditioned cell culture supernatant, and ascites fluid. These results establish the NBSIBA column as a novel affinity chromatography method that utilizes the NBS for selectively purifying antibodies from complex mixtures.

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EXPERIMENTAL SECTION Materials. Indole-3-butyric acid, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), N,N-diisopropylethylamine (DIEA), N-hydroxy-succinimide (NHS), and mouse ascites fluid (clone NS-1) were all purchased from SigmaAldrich (St. Louis, MO). ToyoPearl AF-Amino-650 M resin (87 meq/L amine loading) was purchased from Tosoh Biosciences (King of Prussia, PA). HRP-conjugated goat antihuman IgG Fcγ-specific was purchased from Jackson ImmunoResearch (West Grove, PA). Bovine serum albumin was purchased from EMD Millipore (Billerica, MA). Amplex Red assay kit, Quant-iT PicoGreen dsDNA high-sensitivity assay kit, serum-free hybridoma media (SFM), tissue culture grade L-glutamine, β-mercaptoethanol, and GE Healthcare Life Sciences HiTrap protein G HP columns (1 mL) were purchased from Invitrogen (Grand Island, NY). The thirdgeneration CHO host cell protein (HCP) enzyme-linked immunosorbent assay (ELISA) kit was purposed from Cygnus Technologies (Southport, NC). RMPI-1640 media was 7722

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double-stranded DNA (dsDNA) present in the flow through and elution collected fractions post NBSIBA purification. Conditioned cell culture supernatant and mouse ascites fluid were injected onto the NBSIBA column using the standard purification gradient. An amount of 20 μL of each collected fraction was added to 200 μL of diluted Quant-iT PicoGreen dye reagent (1:200 dilutions in the provided buffer). The solutions were mixed and allowed to incubate for 5 min at room temperature in a 96 well plate protected from light. The amount of dsDNA present in the samples was determined based on dye fluorescence with a 485 nm excitation and 523 nm emission. This fluorescence was converted to nanograms per microliter of dsDNA based on a standard curve. Data represents the means (±SD) of triplicate experiments. Residual Host Cell Protein Content. The HCP content within the load, flow through, and elution fractions was quantified via a third-generation CHO HCP ELISA kit from Cygnus Technologies. The recommended high-sensitivity assay as provided by the manufacturer was followed. Briefly, 100 μL of anti-CHO:HRP matrix was added to each well followed by 50 μL of standards, controls, and samples. Unknown samples were serially diluted to verify signal linearity for accurate quantitation and assay validation. The plate was covered and incubated at room temperature for 2 h. Following incubation the plate was washed with four cycles of ∼350 μL of wash solution. An amount of 100 μL of 3,3′,5,5′ tetramethyl benzidine (TMB) substrate was then added to the wells and incubated for 30 min. To stop the enzymatic reaction 100 μL of stop solution was added. Quantitation was accomplished by reading the absorbance at 450 nm subtracting off the zero standard as a blank.

Millipore Amicon Ultra centrifugal filters. An amount of 20 μL of each concentrated fraction was analyzed on the SEC (size exclusion chromatography) column. Each SEC run was achieved using a 25 min isocratic gradient of 50 mM sodium phosphate buffer at pH 6.8 with 370 mM NaCl and 0.1% Tween20. Determination of Antibody Purity by SDS−PAGE. The purity of antibody in the elution fractions was determined by SDS−PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing conditions, using 10% polyacrylamide gel with a Tris−glycine running buffer (Boston Bioproducts). Sample preparation was done by adding 5 μL of gel loading buffer to 15 μL of concentrated flow through or elution fraction and boiling for 5 min. The quantity of protein run in each lane was normalized within each gel, based on total protein content, but not across gels. Gels were Coomassiestained using Coomassie R-250 (EMD). The antibody purity was determined by densitometric analysis of Coomassie-stained gels using Carestream molecular imaging software (standard ed., v.5.0.2.30). The purity of the product was calculated as the fraction of the total area and intensity equivalent to the IgG bands at 25 and 50 kDa. Determination of Antibody Recovery by ELISA. The flow through and elution fractions collected from the NBSIBA column were diluted 200-fold in a 0.05 M carbonate− bicarbonate buffer pH 9.6 to a final volume of 100 μL and directly adsorbed on a high-binding Costar 96 well plate for 2 h at room temperature. The surface was subsequently blocked with 2.5 g of BSA in 50 mL of phosphate-buffered saline (PBS) pH 7.4 and 0.05% Tween20 for 1 h. Total antibody in each well was determined using an HRP-conjugated secondary antibody and was quantified using an Amplex Red assay kit (570 nm excitation and 592 nm emission). Influence of NaCl, BSA, Conditioned Cell Culture Supernatant, and Ascites Fluid on Antibody Recovery and Purity. To analyze the effect of NaCl and BSA on the NBSIBA column’s performance, samples containing 2.5 mg/mL rituximab in increasing concentrations of NaCl (0, 0.3, 0.6, 1.0, and 1.5 M) or BSA (0, 5, 15, 25 mg/mL) were prepared in 50 mM sodium phosphate buffer at pH 7.0. To analyze the effect of contaminants introduced during typical antibody production/expression, samples containing 1.0 mg/mL rituximab were prepared with hybridoma media SFM, conditioned cell culture supernatant, or mouse ascites fluid. Conditioned cell culture supernatant was obtained by growing CHO-K1 or NCI-H929 myeloma cells (ATCC) for 3 days in F-12K (Kaighn’s modification) or RPMI 1640 media, respectively, containing 10−20% FBS, and L-glutamine. The conditioned media was centrifuged to remove whole cell content. The purchased mouse ascites fluid was collected and centrifuged to remove whole cell content, and 15 mM sodium azide was added as a preservative by the vendor. For analysis of the above-described samples, 20 μL of each sample was injected on the NBSIBA column. Flow through and elution fractions were collected for determination of antibody recovery and purity. Peak integrations were carried out using Agilent Technologies ChemStation LC software. All samples were analyzed by UV absorption at 220 or 280 nm wavelength, and chromatograms have been background-subtracted based on a 20 μL blank injection of equilibration buffer. Sample handling for SEC, SDS−PAGE, and ELISA techniques are outlined in each respective section. Residual Host Cell DNA Content. Quant-iT PicoGreen dsDNA high-sensitivity assay kit was used to quantify the

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RESULTS AND DISCUSSION Selection of Resin and Synthesis of Stationary Phase. In an earlier publication, we extensively characterized the NBS using molecular modeling and showed that it is a highly conserved binding pocket located in the “conserved” region of the variable domain of all antibody isotypes across various species (Figure 1A).26,27 This characterization was achieved by performing a least-squares root-mean-square deviation superposition of all Fab domain crystal structures of >260 immunoglobulins available in the RCSB Protein Data Bank.26 Our analysis revealed that four residues, namely, two tyrosine residues on the light chain [framework region 2 position 42 (FR2, Y42) and FR3, Y103 implementing the IMGT numbering system] and a tyrosine [FR3, Y103] and a tryptophan [junction region, W118] residue on the heavy chain, make up the NBS and are conserved throughout all immunoglobulin isotypes.28 To identify a small-molecule ligand with a high binding affinity and selectivity for the NBS that we can use to capture antibodies in our affinity chromatography method, we performed an in silico screening by docking various small molecules from the ZINC database at the NBS. Our screening revealed IBA as a potential nucleotide analogue that can be used as a capture molecule, with Kd values ranging between 1 and 8 μM depending on the antibody. We have further characterized IBA−IgG interactions and have confirmed that IBA selectively and exclusively associates with the light chain of antibodies.29 Therefore, IBA was chosen for the application described in this study. Resin selection is critical to achieve effective performance from the NBSIBA column; hence, we evaluated several commercially available polymer resins as potential stationary 7723

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Figure 2. (A) Chromatograms demonstrating the effect of antibody concentration on the NBSIBA column’s capture efficiency. (B) Chromatograms demonstrating the effect of changing EQ buffer wash time on retention of rituximab by the NBSIBA column. Sample nos. 1, 2, and 3 corresponds to 5, 10, and 240 min EQ wash times, respectively. (C) NBSIBA column did not display any nonspecific binding for an array of contaminants. (D) Control column packed with acetylated ToyoPearl resin without IBA modification displayed no capture of antibody or contaminants.

following an injection, the column was washed with EQ buffer for 5 min, followed by a gradient of ELS buffer (0−100% over 10 min) where captured antibody is eluted 9 min into the ELS gradient (Figure 2A). Antibody recovery was quantified by peak integration and ELISA. Increasing the amount of antibody injected on the NBSIBA column did not have an effect on capture efficiency and consistently yielded ≥98% antibody recovery. The largest amount of antibody injected on the NBSIBA column was 200 μg of rituximab (20 μL of 10 mg/mL) into a resin volume of 785 μL without any sign of exceeding the column’s antibody capture capacity (Figure 2A). The performance of the NBSIBA column was also validated by trastuzumab, a humanized pharmaceutical antibody (Figure 2A). The ability of the NBSIBA column to retain the captured antibody during EQ wash was also tested at various wash times of 5, 10, and 240 min. The antibody was retained on the column throughout the EQ wash under all conditions, and was eluted consistently 9 min into the ELS gradient, leading to elution times of 14, 19, and 249 min, respectively (Figure 2B). These results suggest that the retention of the antibody on the column is not due to a size exclusion phenomenon. If antibody retention was due to a size exclusion effect of the resin, then the elution time would be independent of the duration of the wash time (Supporting Information Table S-1). Furthermore, when the same amount of antibody (50 μg) was injected using a 50fold dilution (1 mL total volume), the column capture efficiency was not affected (Supporting Information Figure S2). In order to show the specificity of the NBSIBA column in antibody capture, various contaminants were injected to demonstrate that none of the contaminants were retained on

phase candidates for the synthesis. Optimal characteristics of a resin for small-molecule-based affinity purification are (i) limited swelling in high-salt or low-pH aqueous buffers, (ii) monodisperse pore size and resin diameter, (iii) physical stability to nominal pressure drops, (iv) chemically stable polymer backbone, (v) nominal nonspecific protein adsorption, and (vi) highly functionalized surface. ToyoPearl AF-Amino650 M resin is a commonly used resin by many research laboratories, which meets the qualities listed above and provides a suitable and scalable platform from proof of concept to large-scale industrial purification systems. This resin is a methacrylic polymer backbone with hydroxylated aliphatic chains for reduced nonspecific protein adsorption, has 100 nm pore size, and presents primary amine moieties for covalent functionalization with IBA. The stationary phase was prepared by coupling IBA to ToyoPearl resin using an EDC/NHS amide coupling method described previously in the Experimental Section. A cylindrical column with dimensions of 10 mm i.d. and 10 mm length was packed with the IBA-conjugated resin to be used as the NBSIBA column. An acetone pulse was used to determine key column characteristics such as the height equivalent to theoretical plates (HETP) and elution peak asymmetry (As), which were 0.0165 cm and 0.822, respectively (Supporting Information Figure S-1). Antibodies are Selectively Captured and Retained on the NBSIBA Column. To evaluate the NBSIBA column, we used rituximab, a chimeric anti-CD20 pharmaceutical antibody. The column was loaded with 20 μL of the indicated amounts of antibody in 50 mM phosphate buffer (pH 7.0). The antibody was successfully captured on the column using an EQ buffer and was then eluted using a gradient of ELS buffer. Typically, 7724

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of ionic strength in the sample on NBSIBA performance the purification gradient and buffers remained unchanged. Antibody recovery was quantified by comparing the peak integration values, at 280 nm, of the flow through and elution fractions. The integration values from each peak were summed to verify that the entire injected antibody sample was eluted from the column and that the addition of NaCl did not promote irreversible antibody binding to the NBSIBA column. Increasing concentrations of NaCl in the sample injection buffer above 0.3 M NaCl decreased the NBSIBA column’s capture efficiency; we observed only 72% recovery at 1.5 M NaCl (Figure 3). This result was expected given that high salt concentration of the ELS buffer drives antibody elution. Importantly, by simply diluting the high-salt sample with DI water prior to injection, hence reducing the NaCl concentration, the NBSIBA column’s full capture efficiency was recovered (Supporting Information Figure S-3). Effect of BSA Contaminants on Antibody Recovery and Purity. To evaluate the NBSIBA column’s ability to separate antibody from challenging impurities, we contaminated antibody samples with known amounts of BSA at various concentrations prior to injection on the NBSIBA column. We selected BSA as part of our stringent test criteria since it is the major impurity in cell culture supernatants and ascites fluid and is known to aggressively adhere to surfaces through nonspecific interactions. BSA-contaminated antibody samples were injected on the NBSIBA column, and the flow through and elution fractions were collected for further analysis by ELISA, SDS− PAGE, and SEC (Figure 4). On the basis of the ELISA results, no significant changes were observed in the amount of antibody in the elution peak with increasing BSA concentrations. Some low levels of antibody, however, were detectable in the flow through of the BSA-contaminated fractions that ranged from 4% to 7% of the total amount injected. Combined, these results suggest that contaminating the antibody samples with BSA resulted in only a slight reduction in antibody recovery; according to ELISA 93−96% antibody was recovered compared to 98% in a contaminant-free injection (Figure 4B). It is noteworthy that the NBSIBA column performed adequately even at the highest BSA concentration used in these experiments, although such extreme conditions are not representative of the biological fluids antibodies are typically isolated from. The purity of the antibody was analyzed by SDS−PAGE analysis (Figure 4C). A significant increase in the amount of BSA was detectable in the flow through as the BSA contaminant amount in the injection sample increased. However, no BSA was detectable in the elution fractions even at the highest BSA concentration, suggesting that the recovered antibody fractions did not have any BSA impurity. Antibody purity was further analyzed on an SEC column (Figure 4D). Rituximab elutes at 10.3 min, and BSA appears as two peaks eluting at 10.0 and 10.8 min corresponding to BSA dimer and monomer, respectively (Supporting Information Figure S-4A). SEC chromatograms of the flow through fractions from the BSA-contaminated samples displayed the characteristic two peaks corresponding to BSA dimer and monomer. Antibody was not detected in any of the flow through fractions by SEC. SEC chromatograms of the NBSIBA elution peaks demonstrated a single peak eluting at 10.3 min corresponding to the antibody, with no shoulders or broadening around 10.0 or 10.8 min indicating no BSA contamination. Furthermore, superimposing the SEC chroma-

the column. None of the proteins or other biological molecules from these contaminants were retained on the NBSIBA column, and all contaminants eluted within the flow through fraction (0.5−4.0 min) post injection (Figure 2C). These results demonstrate that the column has a high selectivity for antibodies. Efficient capture of pure antibody injected on the column after exposure to the diverse contaminants demonstrates that these contaminants do not have a residual negative impact on the antibody−IBA interaction. To demonstrate that the antibody capture observed with the NBSIBA column was IBA-dependent and was not due to an inherent property of ToyoPearl resin, we tested a control column packed with ToyoPearl resin without IBA modification. As expected, antibody was not retained on the control column, and all injected antibody samples eluted in the flow through (Figure 2D). All together, these results suggest that antibody capture on the NBSIBA column is a result of specific interactions between the immobilized IBA on the resin and the antibody. Furthermore, in a recent publication, we demonstrated that IBA specifically interacted with the antibody Fab domain and photoreaction between IBA and the antibody exclusively yielded conjugation of IBA at the light chain.29 Effect of NaCl Concentration on Antibody Recovery. Efficiency of antibody capture by the NBSIBA column depends on the ionic strength of the sample injection buffer. Therefore, we tested the effect of NaCl concentration in the sample injection buffer on antibody capture efficiency. For this experiment, antibody samples were prepared in phosphate buffer with increasing NaCl concentrations and were injected onto the NBSIBA column (Figure 3A). To purely test the effect

Figure 3. (A) Chromatograms illustrating the effect of NaCl concentration in the injection buffer on antibody capture efficiency by the NBSIBA column. (B) Normalized peak integration values of the flow through (FT) and elution (El) fractions are shown for the above injections. Ratio of load volume to column volume 20/785 μL. 7725

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Figure 4. Effect of BSA contamination on antibody recovery and purity by the NBSIBA column. (A) Chromatograms of rituximab (2.5 mg/mL) premixed with increasing BSA content. (B) ELISA results illustrating percent antibody in the flow through and elution fractions. Data represents the means (±SD) of triplicate experiments. (C) SDS−PAGE analysis showing no BSA contamination in recovered antibody. (D) SEC chromatograms of the flow through (blue traces) and elution fractions (red traces) further establishing the purity of the recovered antibody. Sample nos. 1, 2, and 3 are 5, 15, and 25 mg/mL BSA, respectively, each containing 2.5 mg/mL rituximab.

purified from conditioned cell culture supernatant. This fluorescence was converted to nanograms per microliter of dsDNA by using a standard curve (Supporting Information Figure S-6) and then normalized to antibody content in each fraction. The results demonstrate that DNA flows through the column relatively unimpeded by the IBA or resin backbone leaving a very low level of DNA in the purified antibody elution fraction with a log reduction value (LRV) of 3.14, on par with protein A DNA clearance values.6,30 Residual HCP content in each collected fraction was analyzed via a broadly reactive HCP ELISA assay by using a standard curve (Supporting Information Figure S-7). With a resulting LRV of 1.89 the HCP removal is comparable to that of protein A.6,31 A summary of the HCP and DNA content in the collected fractions is shown in Table 1. These results further support the high level of purity (>95%) that the NBSIBA purification technique can attain. Column Stability and Reusability. The NBSIBA column yielded reproducible results without loss in performance in antibody recovery (100% ± 3.7%) even after 100 injections. Results of representative injections of antibody samples contaminated with conditioned cell culture supernatant (injection 100), BSA (injection 75), and pure antibody (injections 1, 25, and 50) are shown in (Figure 6). Within these 100 cycles of injections, the resin was also exposed to 10 cycles of low pH (25 mM sodium acetate buffer at pH 3.5) to assess the column under chemically harsh conditions, which did not result in any reduction in the column stability or performance. We have also conducted stability experiments of the immobilized IBA on the ToyoPearl resin over the course of 10 cycles of 0.5 M NaOH sterilization exposure and did not observe any leaching of the capture ligand from the resin based

tograms of the elution fractions with a pure antibody sample injection displayed very subtle changes in its composition with 98% conservation of its shape confirming the purity of the isolated antibody. Effects of BSA and salt concentration on antibody recovery and purity are summarized in Supporting Information Table S-2. Effect of Conditioned Cell Culture Supernatant and Ascites Contaminants on Antibody Recovery and Purity. Finally, we evaluated the NBSIBA column’s efficiency to purify antibody from three typical contaminant sources: hybridoma media, conditioned cell culture supernatant, and ascites fluid. Samples of 1 mg/mL rituximab were prepared in these media and were subsequently purified on the NBSIBA column (Figure 5A). The flow through and elution peaks were collected and analyzed for antibody recovery and purity using ELISA and SDS−PAGE. ELISA results suggest no significant loss in antibody recovery from any of the tested contaminant sources (Figure 5B). Furthermore, SDS−PAGE gel results showed that all impurities eluted within the flow through fraction, leaving the elution fraction free of any contaminants with a purity of ≥98% (Figure 5C). A control experiment with protein G was also ran side by side and analyzed in a parallel gel ran under the same conditions. Our results demonstrated comparable level of purity to the standard protein G purification (Figure 5C). A control gel was also run in parallel with only the contaminant sources (Supporting Information Figure S-5). These antibody recovery and purity results are summarized in Supporting Information Table S-3. Host cell DNA removal from the purified antibody was also determined via binding of fluorescent dye to dsDNA present in the load, flow through, and elution fractions of antibody 7726

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Figure 6. Effect of injection number on antibody recovery by the NBSIBA column. (A) Overlaid chromatograms of representative injections of rituximab (2.5 mg/mL) on the NBSIBA column with various protein contaminates over the course of the 100 injections. The flow through portion (0−8 min post injection) was removed from the figure to enhance the clarity of the presentation. (B) Percent antibody recovery based on 280 nm peak integration of the representative injections of rituximab contaminated with conditioned cell culture supernatant (injection 100), BSA (injection 75) as well as pure antibody (injections 1, 25, and 50). “Avg.” represents the mean (±SD) of the five representative injections.

injected on an analytical C18 reversed-phase HPLC column over the course of 48 h. On the basis of elution time, peak elution profile, and peak intensity there was no discernible modification to the capture ligand as a result of exposure to the alkaline sterilization conditions (Supporting Information Figure S-9).

Figure 5. Effect of various sources of contamination on antibody recovery and purity by the NBSIBA column. (A) Chromatograms of rituximab (1 mg/mL) prepared in hybridoma media, conditioned cell culture supernatant, and mouse ascites. (B) ELISA results illustrating percent antibody in the flow through (FT) and elution (El) fractions. Data represents the means (±SD) of triplicate experiments. (C) SDS− PAGE analysis showing no protein contamination in recovered antibody. Sample no. 1 is pure antibody control; sample nos. 2, 3, and 4 are antibody purified from hybridoma media, conditioned cell culture supernatant, and mouse ascites, respectively. A control experiment with protein G was also pursued and ran in a parallel gel under the same conditions.

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CONCLUSION The results presented in this study establish a novel affinity chromatographic strategy that utilizes the NBS for the selective purification of antibodies from complex mixtures. The NBSIBA column consistently yielded >95% antibody recovery with >98% purity during purifications performed with the chimeric monoclonal antibody rituximab, even when challenged with various contaminants such as hybridoma media, conditioned cell culture supernatant, and ascites fluid. Furthermore, the NBSIBA column yielded reproducible results without loss in performance in antibody recovery (100 ± 3.7%) even after 100 injections. A caveat inherent to affinity chromatography is that the column performance is strongly dependent on the affinity of the nucleotide analogue for the NBS, which ranges between Kd = 1 and 8 μM for IBA, depending on the antibody. The four residues that are responsible for forming the NBS are conserved throughout all antibody isotypes and across most species; therefore, we believe that the differences in affinity are most likely due to the variations of the residues that cause a structural shift in the framework region or the residues that make up the

Table 1. DNA and HCP Content of Load and Collected Fractions from Conditioned Cell Culture Supernatant Purification on an NBSIBA Column fractions

HCP (ng/mg mAb)

DNA (ng/mg mAb)

load flow through elution LRV

343,800 8,983,800 4,427 1.89

61,072 463,101 44 3.14

on absorbance readings at 280 nm. The IBA capture molecule itself was also incubated in 0.5 M NaOH and was periodically 7727

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Analytical Chemistry

Article

“lip” of the nucleotide binding site. The accessibility of the binding site (steric hindrance) as well as the polarity and the hydrophobicity of the residues at the lip may contribute to the minor changes observed in the binding affinity of the nucleotide analogue for different antibodies. These minor changes, however, can reflect as an amplified difference in the avidity of the antibody for the multivalent presentation of IBA on the resin and may result in reduced column performance. In this study, we used rituximab, a chimeric antibody, which favorably interacts with IBA (Kd = 1.7 μM), to establish the NBSIBA column’s potential for antibody purification. Implementing the NBSIBA column with antibodies that exhibit a lower monovalent binding affinity to IBA may result in reduced column performance as observed by a reduction in antibody recovery with murine antibodies (Kd = 4−8 μM) (results not shown). We believe that the NBS affinity chromatography method can be further improved by identifying a nucleotide analogue that has a higher affinity for the NBS of all antibody isotypes and species. Furthermore, column performance may also be improved by increasing the density of the capture molecule on the resin surface to enhance the multivalent IBA− antibody interaction, increasing the overall avidity. These studies are currently ongoing in our laboratory to further improve the NBS column such that it can be applied universally to all antibodies. Taken together, the NBSIBA column provides significant potential advantages over current antibody purification methods such as lower production cost, more durable column with increased column lifetime, and no leaching of protein A/G into the purified antibody. Importantly, the NBSIBA column selectively captures only active antibodies since the Fab arms must be properly folded for effective binding to IBA. This eliminates the capture of misfolded, aggregated, and inactive antibody molecules that cannot be differentiated by protein A/ G columns. We expect the NBSIBA column to be of benefit to life-sciences research, as well as have an impact in therapeutic applications by increasing the quality of the purified antibody product while lowering the overall cost associated with antibody production.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 574-631-1429. Fax: 574-631-8366. E-mail: bbilgicer@ nd.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Rituximab and trastuzumab were gifts from Dr. Rudolph Navari at Indiana University School of Medicine. REFERENCES

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dx.doi.org/10.1021/ac300952r | Anal. Chem. 2012, 84, 7721−7728