Development of chemically stable solid phases for the target isolation with reduced nonspecific binding proteins

Poly(methacrylate) matrices for affinity resins were designed and synthesized based on our previous results that nonspecific protein absorption on affinity resins strongly depended on their hydrophobic property. The novel affinity resins bearing FK506 (6a, 6b) captured specific binding protein, FKBP12, with a small amount of nonspecific binding proteins. The amount of nonspecific binding proteins on 6a-6b was much reduced compared to that on commercially available poly(methacrylate) resins, Toyopearl (8), and was almost the same as that on one of the most popular resins, Affigel (9). Interestingly, 6a and 6b could isolate FKBP52 as a specific binding protein as well, although 8 and 9 could not.     

Selective elution of target protein from affinity resins by a simple reductant with a thiol group

We have made a chance discovery of selective elution of a specific binding protein from affinity resins by mixing them with aqueous solutions of a widely used reductant, 2-mercaptoethanol (2ME), under mild conditions. Our studies suggest this phenomenon would be generic, and could be a powerful method for identification of a specific binding protein. We here exhibit the experimental conditions and successful examples in which target proteins of benzensulfonamide and FK506 were selectively eluted from affinity resins bearing these compounds, while non-specific ones remained.     

Application of inline variable pathlength technology for rapid determination of dynamic binding capacity in downstream process development of biopharmaceuticals

Determination of dynamic binding capacity (DBC) for capture purification chromatographic step is usually the first experiment to be performed during downstream process development of biopharmaceuticals. In this work, we investigated the application of inline variable pathlength technology using FlowVPE for rapid determination of DBC on affinity resins for protein capture and proved its comparability with offline titer methods. This work also demonstrated that variable pathlength technology for DBC determination can be successfully applied to different classes of monoclonal antibodies and fusion proteins. This enabled rapid screening of affinity resins and optimization of the capture chromatography step. Hence, use of inline variable pathlength technology eliminated the dependency on offline titer data, traditionally used for DBC determination and accelerated overall process development timelines with less cost.     

Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library.

A screening assay for the detection of RNA-binding proteins was developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem-loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to beta-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA-protein interaction. Parameters influencing the specificity and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA-protein interaction could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10(-8) M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato.     

Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.     

Detection of Rap1A as a yessotoxin binding protein from blood cell membranes

As is the case with other ladder-shaped polyether compounds, yessotoxin is produced by marine dinoflagellate, and possesses various biological activities beside potent toxicity. To gain a better understanding of the molecular mechanism for high affinity between these polyethers and their binding proteins, which accounts for their powerful biological activities, we searched for its binding proteins from human blood cells by using the biotin-conjugate of desulfated YTX as a ligand. By a protein pull-down protocol with use of streptavidin beads, a band of specifically binding proteins was detected in SDS-PAGE. HPLC-tandem mass spectrometry (MS/MS) indicated that Rap 1A, one of Ras superfamily proteins, binds to the YTX-linked resins. Western blotting and surface plasmon resonance experiments further confirmed that Rap1A specifically binds to YTX with the K(D) value around 4 μM.     

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.     

Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion-exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell-shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the pI, and on both sides of the pH scale, the bell-shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI above the pH of the buffer (the 'unadsorbed' fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre-fractionation techniques currently employed in proteomics.     

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size,shape, and salt concentration

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.     

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and V_HH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.     

Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M_r of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.     

Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation

Z-DNA affinity adsorption of an Escherichia coli lysate in the presence of excess B-DNA results in a 1000-fold enrichment for three proteins with apparent molecular weights, on SDS/polyacrylamide gel electrophoresis, of 50,000, 90,000 and 100,000. When retention of these proteins on resins constructed with Z-DNA (Br-poly(dG-dC).poly(dG-dC)) was compared with retention on resins constructed with B-DNA or Br-B-DNA, it was found that approximately 100-fold more of the 50,000 Mr protein, 1000-fold more of the 90,000 Mr protein, and greater than 1000-fold more of the 100,000 Mr protein was retained on the Z-DNA resin. No difference in retention on the B-DNA versus brominated B-DNA resin was found, indicating that the increased retention on the Z-DNA resin was not due to bromination of the Z-DNA. This demonstration of Z-DNA-specific binding in vitro makes these proteins candidates for binding to Z-DNA in vivo. In an effort to determine the function of these proteins we have prepared monoclonal antibodies against each protein and isolated its respective gene. Western blot analysis of lysogens carrying these genes confirms their identity and shows that the complete coding region and promoter for each gene has been cloned.     

A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteins is important for the success of such biochemical approaches. To develop strategies to circumvent this problem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resins bearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein binding was proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated to the CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compounds had CLOGP values greater than 1.5, (amount of tubulin) = 0.73 x CLOGP - 1.1 (n = 7, r = 0.97), and (amount of actin) = 0.42 x CLOGP - 0.79 (n = 7, r = 0.99). On the basis of these studies, we designed a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands to chromatography resins. As predicted by our binding algorithm, introduction of this spacer reduced the amount of nonspecific protein binding in proportion to the number of ethylene glycol units.     

An exclusion mechanism in ion exchange chromatography

Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes.     

Maximizing productivity of chromatography steps for purification of monoclonal antibodies

The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.     

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin.

Various aspects of the interaction of oestrogen-receptor complexes with calf uterine chromatin covalently coupled to cellulose were analysed. Partially purified [3H]oestradiol-receptor complexes were bound to intact, or partially deproteinized, chromatin resins. Proteins were removed from the chromatin-cellulose resins by extraction with high molarities of salt, including NaCl/urea, guanidine hydrochloride and guanidine thiocyanate. After extensive washing to remove the salt, [3H]oestradiol-receptor-complex solutions were added to the resins and the degree of binding was determined. The extent of [3H]oestradiol-receptor-complex binding to chromatin was enhanced by extraction of chromosomal proteins. By varying the molarity of the salt, and consequently the extent of protein removal, it was possible to resolve [3H]oestradiol-receptor-complex binding to guanidine thiocyanate-extracted chromatin into two components. Similarly, [3H]oestradiol-receptor-complex binding to guanidine hydrochloride-treated chromatin included three regions of enhanced binding capacity. The [3H]oestradiol-receptor-chromatin interaction was saturable with respect to both intact and salt-extracted resins. Thus uterine chromatin may contain three or more specific classes of acceptors for the oestrogen-receptor complex.     

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Discovery of binding proteins for a protein target using protein–protein docking‐based virtual screening

Target structure‐based virtual screening, which employs protein‐small molecule docking to identify potential ligands, has been widely used in small‐molecule drug discovery. In the present study, we used a protein–protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all‐to‐all protein–protein docking run on a large dataset was performed. The three‐dimensional rigid docking program SDOCK was used to examine protein–protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z‐score, and convergency of the low‐score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all‐to‐all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor‐α (TNFα), which is a well‐known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top‐ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein–protein docking for the discovery of novel binding proteins for specific protein targets. Proteins 2014; 82:2472–2482. © 2014 Wiley Periodicals, Inc.     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014