Selective displacement chromatography of proteins

In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of alpha-lactalbumin and beta-lactoglobulin A and B has been used for anion-exchange systems; a four-protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on-line monitoring can be readily employed for the selective displacement process, thus facilitating the scale-up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.     

Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

Hydrophobic displacement chromatography of proteins

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.     

One-step affinity purification protocol for human telomerase.

Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of approximately 550 kDa compared to the approximately 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.     

Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting

Carrier-mediated delivery holds great promise for significantly improving the cellular uptake and therefore the therapeutic efficacy of antisense oligonucleotides in vivo. A multivalent carbohydrate recognition motif for the asialoglycoprotein receptor has been designed for tissue- and cell-specific delivery of antisense drugs to parenchymal liver cells. To combine low molecular weight with high receptor affinity, the synthetic ligand contains three galactosyl residues attached to a cholane scaffold via epsilon-aminocapramide linkers. Three-dimensional structural calculations indicate that this unique design provides proper spacing and orientation of the three galactosyl residues to accomplish high affinity binding to the receptor. Covalent conjugation of the bulky carbohydrate cluster to oligonucleotides has been achieved by solid-phase synthesis using low-loaded macroporous resins and optimized synthesis protocols.     

Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

A robotic high‐throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs, and DC‐50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly, selective displacement was found to be conserved for multiple protein pairs, demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities, they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer‐protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer‐protein interactions to other secondary interactions and more selective displacement systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Application of hydrophobic interaction displacement chromatography for an industrial protein purification

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C-terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high-throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary-phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG-3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step-gradient elution process.     

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1_G465R) and CMAC(hOCT1_R488M). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [³H] MMP⁺ (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.     

Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.     

A two step displacement and affinity chromatography purification in lactate dehydrogenase recovery from beef heart extract

Lactate dehydrogenase from beef heart extract was purified by displacement chromatography on a Tris Acryl DEAE. Chondroitin sulphate C, alginate and Eudragits were tried as displacers. Displacement, in conjunction with a subsequent affinity step, gave high purifications and yields. The potential of this operational mode as an early step in protein recovery and the use of L and S forms of Eudragit as cheap readily available and non-toxic displacers has been demonstrated in this work.     

A continuous affinity ultrafiltration process for trypsin purification

A continuous process has been devised and tested for purification of a crude trypsin preparation from pig pancreas. The development was based on the principles of affinity chromatography and Ultrafiltration. Trypsin was selectively attracted by a water-soluble high molecular weight (>100,000) polymer, bearing a potent and specific trypsin inhibitor, m-aminobenzamidine. The trypsin-macroligand complex was then retained by using an appropriate Ultrafiltration membrane, while impurities could pass through. The bound trypsin was eluted by either arginine or benzamidine. The process also featured provision for recirculation of the eluant as well as the macroligand. It was demonstrated that this purification process could purify trypsin from the crude preparation with a yield of 77%, contaminated with only 3% of impurities. For the first time, a serious attempt has been made toward continuous purification of enzymes by the affinity Ultrafiltration technique. Besides a substantial increase in productivity, the affinity polymer could be easily reconditioned and expected to possess a long operative life. Such characteristics undoubtedly will play an important role in reducing the cost of trypsin purification.     

Bovine proacrosin from cauda epididymal sperm: purification, characterization and partial sequencing at N-terminus.

1. In the present study, we isolated the two forms of proacrosin from acid extracts (pH 3.0) of cauda epididymal bovine spermatozoa by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and affinity chromatography on Concanavalin A Sepharose 4B. The overall purification was 13-fold with respect to crude acid acrosomal extract. 2. The apparent molecular weight of the proacrosins determined by SDS-PAGE were 44,000 and 38,000. Both forms have proteinase activity on gelatin-SDS-polyacrylamide gel electrophoretic zymography. 3. The M(r) = 38,000 component was isolated by reverse phase HPLC. Thirty-nine amino acid residues at the N-terminus have about 72 and 77% sequence similarity with boar and human proacrosin, respectively. 4. The amino acid sequence of 14 amino acids at the N-terminus of the high molecular weight component (M(r) = 44,000) was determined after electroblotting on a polyvinylidene difluoride membrane. This portion of the molecule is identical with that of the low molecular weight component. 5. Proacrosin autoactivation followed the sigmoidal activation curve.     

从人脐带血中分离纯化血纤溶酶原

以人血清为原料 ,利用纤溶酶原对L型赖氨酸的高亲和性制备了Lysine -Sepharose4B和Lysine -Agarose ,以亲和层析法从人血浆中提取和纯化血纤溶酶原 (plasminogen ,PGn)。利用聚丙烯酰胺凝胶电泳对其纯度和分子量进行分析 ,结果表明纯化得到的为 92kDa的单一组分的人血纤溶酶原。这种纯化方法的建立为进一步大量制备血管生成抑制素 (angiostatin)奠定了基础。     

Purification of the c-fos enhancer-binding protein.

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.     

Partial purification of rat and human serum factors inhibiting the G1-S transition in rat hepatocytes

A low molecular weight compound, which inhibits the G1-S transition in rat hepatocytes, was purified from rat trypsin-treated serum by DEAE-cellulose chromatography and high-performance liquid chromatography on three different stationary phases. This procedure led to a 34500-fold purification with a 29% yield. Inactivation of the purified material by specific enzymes showed that the inhibitor is a glycopeptide containing a peptide moiety, N-acetylneuraminic acid and galactose residues. Amino acid analyses indicated the possible existence of a pentapeptide structure. The same purification procedure was applied to the corresponding human inhibitor. Inactivation by specific enzymes showed that it is also a glycopeptide.     

Ion-exchange and affinity chromatography costs in alpha-galactosidase purification

The purification of alpha-galactosidase from soybean seeds is a five to six-step procedure consisting of cryoprecipitation, acid precipitation and ammonium sulfate fractionation followed by two or three chromatography steps. The procedures, while not optimized, were carried out in a manner that resulted in 414-515-fold purification, as reported previously. The costs of two purification sequences were compared. In the best case, the preparative-scale costs of stationary phase, reagents, and hardware were $790 per million enzyme units, excluding labor. Stationary phase costs predominated over extraction, chromatography reagent, and eluent costs when the stationary phase is replaced after 10-40 cycles of use. However, if stationary phase life exceeds 50-200 cycles, stationary phase costs become similar in magnitude to eluent and reagent costs. Labor costs, which are process-specific and difficult to estimate, exceed all other costs by a factor of 10-50 at a small scale of operation and constitute a major cost, regardless of scale. This case study provides equations and a frame-work for carrying out a first comparison of costs for multistep purification sequences. Column life, throughput, and scale of operation were found to determine not only the magnitude, but also the relative contributions, of the different components that make up purification costs. This analysis shows that there are major opportunities for reducing purification costs through the development of less expensive stationary phases and the implementation of intelligent process control and automation for process scale chromatography.     

Purification of carbonic anhydrase isoenzymes by high-performance affinity chromatography and hydrophobic interaction chromatography

Isoenzymes of carbonic anhydrase were purified by a combination of affinity chromatography and hydrophobic interaction chromatography. Immobilization of sulfonamides on an epoxy-activated support provided a stationary phase for affinity chromatography which was stable to hydrolysis by carbonic anhydrase. A first purification step allowed the isolation of enzymes directly from homogenates of human erythrocytes and rat stomach. Without any further preparation, except the addition of ammonium sulfate to the eluate from affinity chromatography, the isoenzymes could be separated by hydrophobic interaction chromatography with very high recovery of protein and retention of enzymatic activity.     

Polymer displacement/shielding in protein chromatography

An overview of different applications of polymer interactions with ion-exchange and dye-affinity chromatographic matrices is presented here. The strength of interaction between the ligand and the polymer plays a crucial role in deciding the mode of chromatographic application. Charged, non-ionic and thermosensitive polymers such as poly(ethylene imine), poly(N-vinyl pyrrolidone) and poly(vinyl caprolactam) respectively, show different degrees of interaction with the dye molecules in dye ligand chromatography. Polymers, with their ability of multipoint and hence strong attachment to the chromatographic matrices, were used as efficient displacers in displacement chromatography. The polymer displacement resulted in better recoveries and sharper elution profiles than traditional salt elutions. The globule–coil transition of the thermosensitive reversible soluble–insoluble polymer, poly(vinyl caprolactam), can be exploited in dye-affinity columns for the temperature induced displacement of the bound protein. In another situation, prior to the column chromatography of crude protein extract, polymers formed complexes with the dye matrix and “shielded” the column. The polymer shielding decreased the nonspecific interactions without affecting the specific interactions of the target protein to the dye matrix.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.     

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.