A fluidized-bed-riser adsorption system for continuous bioproduct recovery from crude feedstock

In this work, a novel technique for continuous purification of biologics from a crude feedstock is demonstrated with equipment referred to as Fluidized Bed Adsorption System (FBRAS). The development and validation of such unit operations were performed utilizing lysozyme as a model protein and Relisorb™ SP405/EB as a carrier. The performance of FBRAS to carry out combined clarification and purification was evaluated by capturing of antifungal peptides directly from the lysed broth. The novel technique reduced the number of process unit operations from six to three without having an impact on purity. Overall productivity increased by 250% in comparison to the existing downstream processing routine.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

LdARL-1 His-tagged recombinant protein: purification by immobilized metal affinity expanded bed adsorption

Previously we have cloned three ADP-ribosylation factor-like (ARL) genes from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, LdARL-1. LdARL-3A was previously purified as an active native form, which was able to bind GTP in vitro. In this paper, we have performed the production and the purification of Histidine-tagged (His-tagged) LdARL-1 recombinant protein by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology. This protein was purified with more than 95% purity and could be successfully used for GTP-binding assay.     

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

内含肽在构建亲和纯化系统中的应用

内含肽是前体未成熟蛋白中的一段具有自我剪接功能的多肽链,在蛋白质纯化、蛋白质连接、环肽制备、蛋白标记以及生物传感器等方面广泛应用。本文综述了内含肽应用于蛋白质亲和纯化的发展历程,分别对层析型和非层析型内含肽纯化体系进行了分析和讨论,并总结了对控制内含肽断裂反应所进行的研究,为进一步改善内含肽介导蛋白质纯化提供依据和线索。     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme

Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100–300 μm, mean pore size: 1–3 μm, pore volume: 57–59%, and bulk density: ～1,438 kg/m³) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high‐throughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads‐CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high‐throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m³ h) and specific activity (2.2 × 10⁵ U/mg) were higher than that obtained with traditional particulate resins. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

An efficient system for high-level expression and easy purification of authentic recombinant proteins

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.     

重组N-乙酰鸟氨酸脱乙酰基酶的高效表达与纯化

利用质粒pET-22b( )为表达载体,成功构建了高效表达N-乙酰鸟氨酸脱乙酰基酶的基因工程菌BL21-pET-22b( )-argE。研究了该菌的最适超声破壁条件及硫酸铵分级沉淀的最适范围,并以金属螯合亲和层析法纯化含有6-His-Tag的目的蛋白。结果表明:26℃诱导表达的菌体,最佳超声破碎条件为功率200 W,超声时间为15min;目的蛋白主要存在于40%~50%硫酸铵沉淀中。通过Ni-NTA亲和层析柱纯化目的蛋白,可以达到SDS-PAGE电泳纯,并显示单亚基相对分子质量为43 000。纯化倍数为139倍,回收率为11.1%。     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

Temperature-triggered purification of antibodies

In this article the unique capability of elastin-like protein (ELP) to reversibly precipitate was combined with the high affinity and specificity of antibody-binding domains such as Protein G, Protein L, or Protein LG as a general method for antibody purification that combines in a unique manner the simplicity and robustness of temperature-triggered precipitation with the selectivity of affinity interactions. In a single precipitation step, antibodies derived from different sources (animal sera or hybridoma cell cultures) were selectively recovered by a simple temperature trigger. Due to the versatility of the binding ligands toward different classes of antibodies, we believe that this technology will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.     

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.     

小鼠pdd87基因在大肠杆菌中的表达与纯化

利用PCR和基因重组技术构建了三个小鼠pdd87基因的原核表达质粒：表达全长cDNA的pET-28a-pdd87质粒;表达PDD87C端404个氨基酸的pET-28a-pdd87-404质粒;表达全长cDNA的pMXB10-pdd87质粒。经IPTG诱导后三种质粒都得到表达。pET-28a-pdd87质粒和pET-28a-pdd87-404质粒表达的蛋白经His6亲和层析纯化后分别获得了带His标签的PDD87蛋白和含C端404个氨基酸的蛋白。pMXB10-pdd87质粒表达的蛋白经几丁质柱亲和层析纯化后获得了纯的PDD87蛋白。     

Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid-liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase-loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K(2)HPO(4) with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6-fold with a recovery of 86%, and lipase B by 1.5-fold with a recovery of 76%.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.     

Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Purification of transforming growth factor type e

Transforming growth factor type e (TGFe) is a heat- and acid-stable polypeptide with an apparent molecular weight of 22,000, which stimulates the proliferation of certain epithelial and mesenchymal cells in monolayer and soft agar. TGFe has been purified to homogeneity. Initial acid-ethanol extraction of bovine kidney was followed by batch ion-exchange chromatography utilizing Bio Rex 70 resin. The activity eluted from the Bio Rex 70 resin was concentrated and diafiltered using an Amicon concentrator equipped with an S1Y10 spiral membrane, then was further purified by Bio-Gel P-60 molecular sieve chromatography. Active fractions from molecular sieve chromatography were pooled and purified by heparin-Sepharose affinity chromatography, followed by reverse-phase high-performance liquid chromatography using a microbore C-8 column. The final purification step involved electro-elution of TGFe separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purity of TGFe was assessed to be greater than 90%.