Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1 M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl₂ at 0.1–1 M and ethanol at 5–20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.     

Improved performance of column chromatography by arginine: dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.     

Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases

Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).     

Utilization of Arg-elution method for FLAG-tag based chromatography

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein–protein or protein–ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5–1 M arginine at pH 3.5–4.4, as reported in this paper.     

The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography

Arginine is effective in suppressing aggregation of proteins and may be beneficial to be included during purification processes. We have shown that arginine reduces non-specific protein binding in gel permeation chromatography and facilitates elution of antibodies from Protein-A columns. Here we have examined the effects of arginine on binding and elution of the proteins during hydrophobic interaction (HIC) and ion- exchange chromatographies (IEC) using recombinant monoclonal antibodies (mAbs) and human interleukin-6. In the case of HIC, the proteins were bound to a phenyl-Sepharose column in the presence of ammonium sulfate (AS) with or without arginine and eluted with a descending concentration of AS. While use of 1 M AS in the loading buffer resulted in complete binding of the mAb, inclusion of 1 M arginine in loading and equilibration buffer, only when using low-substituted phenyl-Sepharose, resulted in weaker binding of the proteins. While decreasing AS concentration to 0.75 M resulted in partial elution of the mAB, elution was facilitated with inclusion of 0.5-1 M arginine. In the case of IEC, arginine was included in the loading samples. Inclusion of arginine during binding to the IEC columns resulted in a greater recovery and less aggregation even when elution was done in the absence of arginine. These results indicate that arginine enhances elution of proteins bound to the resin, suggesting its effectiveness as a solvent for elution in HIC and IEC.     

Alternative downstream processes for production of antibodies and antibody fragments

Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography

Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl₂. These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6–1.0 M arginine at pH 3.0–3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1536–1541, 2015     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

Role of arginine in protein refolding, solubilization, and purification

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.     

Citrate synthases from germinating castor bean seeds – I. Purification and properties

The mitochondrial and glyoxysomal citrate synthase (EC 4.1.3.7) from the endosperm of germinating castor beans ( Ricinus communis L., type Sanzibaricnsis) were purified to a final specific activity of 76 and 78 U (mg protein)⁻¹, respectively. Both citrate synthases could be bound to ATP-Sepharose. However, only the mitochondrial enzyme could be eluted by either 100 μ M oxaloacetate or 100 μ M coenzyme A (indicative of affinity chromatography), while the glyoxysomal enzyme was only eluted by 0.5 M KCI (indicative of ion-exchange chromatography). Many properties of the two isoenzymes were similar including the pH dependence and temperature dependence of activity, the pH stability, and the inactivation of the enzyme at elevated temperatures. The most pronounced differences between the two citrate synthases were the isolelectric points of pH 5.9 for the mitochondrial and of pH 9.1 for the glyoxysomal enzyme. Both citrate synthases are dimers in the native form with a molecular weight of 95000 each, as determined by gel filtration on Sepharose CL-6B and by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. However, the glyoxysomal citrate synthase existed also as a tetramer with a molecular weight of 200000 in the presence of 10 m M MgCl₂.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Removal of copper, lead, and zinc from contaminated water by saltbush biomass: analysis of the optimum binding, stripping, and binding mechanism

Experiments performed on the Cu(II), Pb(II), and Zn(II) binding by saltbush biomass (Atriplex canescens) showed that the metal binding increased as pH increased from 2.0 to 5.0. The highest amounts of Cu, Pb, and Zn bound by the native biomass varied from 48-89%, 89-94%, and 65-73%, respectively. The hydrolyzed biomass bound similar amount of Pb and 50% more Cu and Zn than the native. The esterified biomass had a lower binding capacity than native; however, esterified flowers bound 45% more Cu at pH 2.0 than native flowers. The optimum binding time was 10 min or less. More than 60% of the bound Cu was recovered using 0.1 mM HCl, while more than 90% of Pb was recovered with either HCl or sodium citrate at 0.1 mM. For Zn, 0.1 mM sodium citrate allowed the recovery of 75%. Results indicated that carboxyl groups participate in the Cu, Pb, and Zn binding.     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

Reusability of avidin-biotinylated immunoglobulin Y columns in immunoaffinity chromatography

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatography was examined by repeated use and regeneration. An enzyme-linked immunosorbent assay-elution assay using CovaLink NH microtiter plates was used to find the optimal conditions for regeneration of columns. Actigel avidin-biotinylated IgY column retained about 90% of its initial IgG binding capacity after 50 cycles, with 0.1 M glycine-HCl buffer, pH 2.8, as eluent, requiring no regeneration. However, IgG binding capacity of UltraLink avidin-biotinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycles, respectively, with the commercial eluent, Actisep. Results from the CovaLink NH system agreed with those from UltraLink avidin-biotinylated IgY columns. The UltraLink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine-HCl, pH 1.6, to dissociate biotinylated IgY antibodies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY to the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidin-biotinylated IgY columns are reusable with or without regeneration depending on the avidin-immobilized matrix.     

Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.