Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

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     

A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing

Available protocols for stripping antibodies from immunoblots involve the use of sodium dodecyl sulfate (SDS) or low-pH buffers. SDS was shown to remove transferred proteins from membranes, and low-pH buffer was shown to inefficiently strip off antibodies. A solution containing 6 M guanidine hydrochloride, 0.2% nondenaturing detergent, and a reducing agent can rapidly strip off tightly bound antibodies from aged polyvinylidene fluoride (PVDF) immunoblots at room temperature without removing significant amounts of transferred protein.     

Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining

Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10^-9 K_D). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.     

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification,Characterization, and Mitigation

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019     

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019     

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.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.     

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid‐enveloped viruses. However, some proteins have limited stability at low pH or in the presence of the detergents, and alternative techniques for achieving the inactivation of enveloped viruses would be beneficial. We present here an alternative and novel approach for the rapid inactivation of enveloped viruses using pH‐neutral buffer solutions containing arginine. The implementation of this approach in a monoclonal antibody or Fc‐fusion protein purification process is described and illustrated with several different therapeutic proteins. The use of the neutral pH arginine solution was able to effectively inactivate two enveloped model viruses, with no measurable effect on the product quality of the investigated proteins. Thus, the use of pH‐neutral arginine containing buffer solutions provides an alternative means of virus inactivation where other forms of virus inactivation, such as low pH and/or solvent/detergent treatments are not possible or undesirable due to protein stability limitations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:108–112, 2014     

A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.     

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.     

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography–mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.     

Purification and structural characterization of recombinant rat gamma-interferon from Escherichia coli

An efficient method has been developed for the purification of recombinant rat gamma-interferon (rat rIFN-gamma). The procedure involves extraction of the Escherichia coli cell paste with 6 M guanidine-HCl (GuHCl), adsorption of the rat rIFN-gamma onto C8 alkyl-bonded silica, and elution with 50% propanol. The protein is essentially pure at this step, but is quantitatively precipitated by threefold dilution with aqueous buffer at pH 8.5. The precipitate is then dissolved with 6 M GuHCl in a buffer containing 0.05%. Tween-80 to about 0.3 mg/ml and dialyzed against the same buffer. The rat rIFN-gamma, which remains soluble on dialysis is again precipitated by dialysis against ammonium sulfate at 80% saturation. This final precipitate is readily soluble in 0.1 M ammonium acetate buffer, pH 8.5. The preparation is fully active and possesses a specific activity of 2-6 X 10(6) units/mg. The recoveries ranged from 50 to 85% in several experiments. The sequence of 20 amino acid residues from the NH2-terminus of the protein was determined using an automated sequencer and was found to agree with that deduced from the cDNA sequence.     

Using high throughput screening to define virus clearance by chromatography resins

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process‐related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non‐infectious retrovirus‐like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96‐well batch‐binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow? (QSFF) and Capto adhere?, a mixed mode resin. QSFF batch‐binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product‐specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96‐well batch‐binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins. Biotechnol. Bioeng. 2013; 110: 1984–1994. © 2013 Wiley Periodicals, Inc.     

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Single‐chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel?), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere?). The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere? in binding‐mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere? below its theoretical pI, while the majority of HCPs were in the flow‐through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Analytical grade C-phycocyanin obtained by a single-step purification process

C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step.     

Optimum Conditions for Indoleacetic Acid Oxidase Extraction from Betula Leaves

This report deals with total extraction and activation of soluble indoleacetic acid oxidase from Betula alleghaniensis leaves as affected by different buffers, varying pH, phenol binder, detergent, plus volume and time parameters. For all buffers and pH levels tested, only tris pH 8 gave a high activity. This result was not a pH effect, since a wide-range, citrate-phosphate buffer at pH 8 gave a very low activity. Addition of a neutral detergent, Triton X-100, to all buffers gave considerable activity in every case. Most activity with Triton X-100 occurred at pH 6 and least at pH 8 regardless of buffer composition. A phenol binder, polyvinylpyrollidone, increased activity also, but less than the detergent Triton X-100. Both of these compounds in combination gave an additive effect and the highest measure of enzyme activity. Further increases in measurable indoleacetic acid oxidase activity were obtained by using the best combination of these factors to determine the optium tissue: buffer ratio and optimum soaking time. Increases in activity of 70 and 60%, respectively, were achieved.     

Performance optimization of continuous countercurrent tangential chromatography for antibody capture

Recent studies have demonstrated that continuous countercurrent tangential chromatography (CCTC) can effectively purify monoclonal antibodies from clarified cell culture fluid. CCTC has the potential to overcome many of the limitations of conventional packed bed protein A chromatography. This paper explores the optimization of CCTC in terms of product yield, impurity removal, overall productivity, and buffer usage. Modeling was based on data from bench‐scale process development and CCTC experiments for protein A capture of two clarified Chinese Hamster Ovary cell culture feedstocks containing monoclonal antibodies provided by industrial partners. The impact of resin binding capacity and kinetics, as well as staging strategy and buffer recycling, was assessed. It was found that optimal staging in the binding step provides better yield and increases overall system productivity by 8–16%. Utilization of higher number of stages in the wash and elution steps can lead to significant decreases in buffer usage (～40% reduction) as well as increased removal of impurities (～2 log greater removal). Further reductions in buffer usage can be obtained by recycling of buffer in the wash and regeneration steps (～35%). Preliminary results with smaller particle size resins show that the productivity of the CCTC system can be increased by 2.5‐fold up to 190 g of mAb/L of resin/hr due to the reduction in mass transfer limitations in the binding step. These results provide a solid framework for designing and optimizing CCTC technology for capture applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:430–439, 2016