人血清白蛋白纯化技术研究进展

本文综述了国内外关于血浆人血清白蛋白（pHSA）和基因重组人血清白蛋白（rHSA）分离纯化的进展和发展趋势。以冷乙醇沉淀为主的pHSA分离方法仍是目前多数工业采用的工艺,但近年来发展的离子交换色谱、凝胶过滤色谱、亲和色谱等多种色谱分离技术具有自动化程度高、生产周期短、更符合GMP等特点,正在逐步取代传统的冷乙醇沉淀法。当前用基因工程技术表达的人血清白蛋白对分离纯化提出了新的挑战。为获得高纯度、安全、稳定的产品,各种色谱分离技术得到更多的应用,其中扩张床离子交换色谱可以省去离心、过滤等传统固液分离操作。尽管rHSA的纯化技术已有一定的发展,但纯化过程仍需进一步优化以提高产品纯度及收率。     

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.     

Recovery of Nuclease Produced by Lactococcus Lactis Using Expanded Bed Ion Exchange Chromatography

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml⁻¹. The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield.     

Fermentation and isolation of epidermin,a lanthionine containing polypeptide antibiotic from Staphylococcus epidermidis

Summary Staphylococcus epidermidis TÜ 3298/DSM 3095 produces epidermin, a basic 21-residue peptide-amide antibiotic active against aerobic and anaerobic Gram-positive bacteria. Fermentations were performed by batch and feeding processes up to the 2001 scale. Highest yields were obtained when the first purification step was integrated into the fermentation process by on-line adsorption of the antibiotic. Isolation and purification by adsorption chromatography and ion exchange chromatography were performed batchwise.     

Routine manufacture of recombinant proteins using expanded bed adsorption chromatography

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.     

Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae

The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.     

Development of a process for large-scale chromatographic purification of an alginate lyase from Klebsiella pneumoniae

Summary A process for purification of an alginate lyase, produced extracellularly by fermentation of Klebsiella pneumoniae, has been developed. The process includes two chromatographic steps and is well suited to large-scale operation. By hydrophobic interaction chromatography on Phenyl-Sepharose FF, followed by anion exchange chromatography on Q-Sepharose FF in a negative mode, the specific activity was increased from 0.09 units (U) mg ^–1 to more than 50 U mg^–1. Due to an extremely low product concentration in the fermentation broth, and large amounts of contaminating proteins, the chromatographic adsorbents had low capacities with respect to alginate lyase. By adsorption on the cation exchanger S-Sepharose FF, the capacity was so low that the enzyme could not be concentrated. The binding capacity of Phenyl-Sepharose FF was approximately 20-fold higher, and a three to tenfold concentration was obtained. The first stage of the process, hydrophobic interaction chromatography, has been applied to the isolation of alginate lyase from fermentation batches of 180 l. Several runs have resulted in a purified product with an average quantity of 30 000–35 000 U per fermentation, and an average specific activity of 4.5 U mg^–1. Although the raw material employed in this work has been particularly unfavourable, the process developed will also be applicable to raw materials with higher product concentrations. Offprint requests to: I. M. Aasen     

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization

Onconase, an RNAse extracted from embryos of the Northern leopard frog ( Rana pipiens ), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an α-mating factor (α-MF)-prepro secretion signal, and an α-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L⁻¹ for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.     

绵毛嗜热丝孢菌木聚糖酶的纯化与性质

研究了绵毛嗜热丝孢菌Thermomyces lanuginosus W205胞外木聚糖酶的纯化与性质。粗酶液经硫酸铵沉淀和Q-Sepharose FF离子交换层析即可得到电泳纯木聚糖酶,回收率为46.6%,比酶活为1396.9U/mg。该酶的最适pH和最适温度分别为pH7.0和75℃,pH稳定范围为5.5-10.8,70℃处理30min残存酶活在70%以上。薄层层析结果显示该酶水解桦木木聚糖的主要产物是木二糖和木三糖,并且能够通过转糖苷作用将木三糖转化为木二糖。该木聚糖酶易于纯化并且具有较宽的pH稳定性及良好的热稳定性,具有较大的潜在工业应用价值。     

人干扰素α—2b生产工艺的研究

人干扰素α-2b的简易、高效生产工艺研究,包括高效率、小型化的发酵技术,不用单克隆抗体亲和层析的纯化工序,以及大肠杆菌表达的人干扰素α-2b包涵体蛋白的天然构型复性等问题,从10L基因工程大肠杆菌发酵液所得1000g菌体中得人干扰素α-2b约500mg,效价为1×10~8u/mg,设备投资少,生产成本低,产品表现了高纯度、高效价。适宜大量生产,为广大人民群众提供廉价高质量药品创造了条件。     

Recovery of potassium clavulanate from fermentation broth by ion exchange chromatography and desalting electrodialysis

Ion exchange chromatography (IEC) and desalting electrodialysis (DSED) processes were developed for the recovery and purification of potassium clavulanate (KCA) from fermentation broth. A strong anion exchanger, Amberlite IRA 400 resin, a potassium acetate solution as equilibrium buffer, and a potassium chloride (KCl) solution as elution buffer were used for the recovery of KCA in IEC. In order to determine optimal operating conditions, the effects of various operating parameters such as equilibrium buffer pH and concentration, elution buffer concentration, gradient length, and volumetric flow rate on KCA recovery and by-product removal were investigated using a simulated fermentation broth. In the subsequent step of DSED, employing cation (Neocepta CMS, Tokuyama, Japan) and anion (Neocepta ACS, Tokuyama, Japan) exchange membranes were carried out to remove KCl that existed in a large amount in the ion exchanged solution. The effects of operation voltage and feed composition on the performance of DSED were investigated. Based on the operating conditions determined above, IEC and DSED were applied in sequence to an ultrafiltered fermentation broth. Almost complete removal of KCl was possible with no significant loss of KCA, although the KCA recovery was slightly lower than that with the simulated fermentation broth. Based on this observation, it was concluded that IEC and DESD could be an effective process combination for the recovery of KCA from fermentation broth.     

Strategies for improving production and purification of a recombinant protein: rP30 of Toxoplasma gondii expressed in the yeast Schizosaccharomyces pombe

Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.     

High-level secretory production of recombinant human platelet-derived growth factor-BB by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under the non-selective conditions

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) was produced by Saccharomyces cerevisiae. A two-stage cultivation strategy with mixture of glucose and galactose was developed to enhance rhPDGF-BB production, with its concentration being 32 mg/l fermentation broth under optimal conditions: corn steep powder as nitrogen source, 2 g/1 of glucose concentration at the beginning of induction phase, a pH of 5.0 and 6.5 for cell growth and rhPDGF-BB accumulation. The purification process consisted of yeast supernatant ultrafiltration followed by ion exchange chromatography and molecular sieve, and the recovery of rhPDGF-BB was estimated to be 20.28%. Biological activity of the purified rhPDGF-BB was 3.05 ng/ml. Published in Russain in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 2, pp. 176–180. The article is published in the original.     

Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.     

Expression and purification of active recombinant human bikunin in Pichia pastoris

Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification procedure. The cDNA encoding human bikunin was cloned by PCR and inserted into the expression vector pPICZαC. After expressed in shake flask, rh-bikunin was produced in an 80-L fermenter and purified by cation exchange chromatography and reverse phase chromatography. The rh-bikunin was active by trypsin inhibition test. The final expression levels were 55 mg/L and we got totally 1.44 g (5600 inhibitor units/mg) of purified rh-bikunin (purity is 95%) from 40 L of fermentation broth. The rh-bikunin consists of two forms with molecular masses of 24 and 21 kDa, respectively. Both forms were immunoreactive by Western blotting and N-terminals were correctly processed by amino-terminal sequencing. This study provided a new method for expression and purification of active rh-bikunin.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.     

High throughput purification of recombinant human growth hormone using radial flow chromatography

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 μm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.     

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two‐step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP‐captured material in batch mode. Using multi‐modal anion exchange in bind‐elute mode, mAb was produced consistently within the purity specification. The benchmark was a state‐of‐the‐art 3‐step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2‐step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase. Biotechnol. Bioeng. 2010;107: 974–984. © 2010 Wiley Periodicals, Inc.     

Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.     

Antimicrobial biosurfactants from marine Bacillus circulans: extracellular synthesis and purification

Aims:  To purify the biosurfactant produced by a marine Bacillus circulans strain and evaluate the improvement in surface and antimicrobial activities.
Methods and Results:  The study of biosurfactant production by B. circulans was carried out in glucose mineral salts (GMS) medium using high performance thin layer chromatography (HPTLC) for quantitative estimation. The biosurfactant production by this strain was found to be growth-associated showing maximum biosurfactant accumulation at 26 h of fermentation. The crude biosurfactants were purified using gel filtration chromatography with Sephadex^® G-50 matrix. The purification attained by employing this technique was evident from UV–visible spectroscopy and TLC analysis of crude and purified biosurfactants. The purified biosurfactants showed an increase in surface activity and a decrease in critical micelle concentration values. The antimicrobial action of the biosurfactants was also enhanced after purification.
Conclusions:  The marine B. circulans used in this study produced biosurfactants in a growth-associated manner. High degree of purification could be obtained by using gel filtration chromatography. The purified biosurfactants showed enhanced surface and antimicrobial activities.
Significance and Impact of the Study:  The antimicrobial biosurfactant produced by B. circulans could be effectively purified using gel filtration and can serve as new potential drugs in antimicrobial chemotherapy.