Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography

The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.     

Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification

The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.     

Development of a novel affinity chromatography resin for platform purification of lambda fabs

Antigen‐binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1311–1318, 2014     

Concanavalin a affinity chromatography for efficient baculovirus purification

Baculovirus has emerged as a novel gene delivery and vaccine vector, and the demand for purified baculovirus is rising due to the increasing in vivo applications. Since the baculoviral envelope protein gp64 is a glycoprotein, we aimed to develop a concanavalin A (Con A) chromatography process, which harnessed the possible affinity interaction between gp64 and Con A, for simple and effective baculovirus purification. Throughout the purification process the virus stability and recovery were assessed by quantifying the virus transducing titers [TT, defined as transducing units (TU) per milliliter] and viral particles (VP). We found that baculovirus stability was sensitive to buffer conditions and diafiltration with a tangential flow filtration system LabScale using 300 K membranes yielded recoveries of ≈75% in TT and 82% in VP. The diafiltered baculovirus strongly bound to the Con A column as evidenced by the low virus losses to the flow through and wash fractions. The wash steps eliminated >99% of protein impurities and elution with 0.6 M α‐D ‐methylmannoside at room temperature led to the recoveries of ≈16% in VP and ≈15.3% in TU. The resultant VP/TU ratio was as low as 41.4, attesting the high quality of the purified virus. Further elution with 1 M α‐D ‐methylmannoside recovered another 6% virus TU, yielding a cumulative recovery of ≈21.3% in TU. These data demonstrated for the first time that Con A chromatography is suitable for baculovirus purification, and may be used for the purification of other viruses with surface glycoproteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Medium format enhancement: design of an immobilized heparin affinity media for industrial bioprocessing

Affinity chromatography is one of the most powerful and selective separation methods available. Recently, affinity methods are being incorporated into industrial processes with some frequency. One of the reasons for this is that affinity media robust enough for industrial bioprocessing are now available. By robust is meant that the media meet stringent requirements for cleanability, sanitization, physical and chemical stability, regulatory and technical support, batch-to-batch reproducibility and reliability of supply. This paper describes a medium format enhancement program to adapt a widely known group-specific affinity medium, Heparin Sepharose( CL6B, to the requirements for industrial bioprocessing. The new medium, Heparin Sepharose( 6 Fast Flow was designed for the recovery of antithrombin 3 (AT3) at industrial scale. The medium is based upon a highly cross-linked 6% agarose, which is produced in very large scale and is familiar to regulatory agencies. The ligand, heparin, is attached to the matrix by a reductive amination chemistry. The resulting linkage is stable in 0.1 N NaOH for 150 h, showing no decrease in AT3 binding affinity at that time. Heparin has a broad biological functionality and thus is useful chromatographically for the purification of a number of proteins which have an affinity for heparin. Heparin, as a complex sugar, is also a highly charged polyanion and thus has interesting ion-exchange properties. Because of its broad applicability to a number of purification problems, immobilized heparin is a useful case study in medium format enhancement. © 1997 John Wiley & Sons, Ltd.     

串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。     

A new procedure for the purification of spinach leaf photosynthetic fructose-1,6-bisphosphatase by affinity chromatography on mercaptoethylamine-Sepharose

A new purification procedure for spinach leaf fructose-1,6-bisphosphatase is proposed, which includes the use of affinity chromatography on mercaptoethylamine-Sepharose. A homogeneous preparation of the enzyme can be obtained in 48 hr, with a specific activity of 67 U/mg and a yield of 23%. The method may also be useful for the purification of other thioredoxin-activated chloroplast enzymes.     

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.     

Complementation cell lines for viral vectors to be used in gene therapy

Viral vectors provide a highly efficient method for the transfer of foreign genes into a variety of quiescent or dividing eukaryotic cells from many animal origins. While recombinant vectors derived from an increasing number of mammalian viruses (herpes simplex virus, autonomous and non-autonomous parvoviruses, poxviruses, retroviruses, adenoviruses available today, vectors based on murine retroviruses and human adenoviruses constitute preferential candidates for the delivery of marker or therapeutic genes into human somatic cells. The availability of such vectors has made possible the recent transition of human gene therapy from laboratory benches to clinical settings. Most current recombinant vectors have been generated by deleting essential viral genes in order to make space available for the introduction of passenger genes. Such vectors are therefore unable to replicate in the absence of these critical gene products and their production relies on the development of stable complementation cell lines providingin trans the missing viral functions. Although complementation (or packaging) cell lines are available for both adenovirus and retrovirus vectors, their respective drawbacks still limit their use to research applications and phase I clinical trials. The future success or failure of human gene therapy will therefore rely on the production of improved generations of packaging cell lines that can produce safer and more efficient vectors which are fully adapted to large scale production and clinical applications.     

Development of a novel purification method for AAV vectors using tangential flow filtration

Adeno-associated virus (AAV) vector can efficiently transduce therapeutic genes in various tissue types with less side effects; however, owing to complex multistep processes during manufacture, there have been surges in the pricing of recently approved AAV vector-based gene therapy products. This study aimed to develop a simple and efficient method for high-quality purification of AAV vector via tangential flow filtration (TFF), which is commonly used for concentration and diafiltration of solutions during AAV vector purification. We established a novel purification method using TFF and surfactants. Treatment with two classes of surfactants (anionic and zwitterionic) successfully inhibited the aggregation of residual proteins separated from the AAV vector in the crude product by TFF, obtaining a clearance of 99.5% residual proteins. Infectivity of the AAV vector purified using the new method was confirmed both in vitro and in vivo, and no remarkable inflammation or tissue damage was observed in mouse skeletal muscle after local administration. Overall, our proposed method could be used to establish a platform for the purification of AAV vector.     

Single step immobilized metal ion affinity precipitation/chromatography based procedures for purification of concanavalin A and Cajanus cajan mannose-specific lectin

Concanavalin A and a mannose-specific lectin could be precipitated specifically from extracts of jack bean and Cajanus cajan seeds, respectively, using metal charged EGTA. Single step purification of the lectins was also possible using iminodiacetic acid-Sepharose charged with metal ions. Nondenaturing electrophoresis in polyacrylamide gel and that performed in presence of SDS ascertained homogeneity of the isolated lectins. The migration behavior of the purified lectins was comparable with those of the lectins purified using alternative procedures.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 mol ml^–1, showed a maximum capacity of 9.1 mg Mab ml^–1 and a dynamic capacity of 3.9 mg Mab ml^–1. A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: The performance of PEGylated protein A

Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or “PEGylation” can potentially improve selectivity by sterically suppressing non‐specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media‐associated non‐specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono‐PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media‐associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two‐ to three‐fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass‐transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1364–1379, 2014     

Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography

We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.     

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Using amphotropic retrovirus stocks produced by TELCeB6-A cells that encode the Escherichia coli lacZ gene, we found that complexation with chondroitin sulfate C (CSC) and Polybrene (PB) is an effective means to purify retrovirus. Virus stocks contained high levels of inhibitory activity that blocked amphotropic, but not ecotropic, retrovirus transduction. When virus stocks were brought to 80 microg/mL each of CSC and PB, complexes of CSC and PB formed. These complexes incorporated more than 70% of the virus particles but less than 0.4% of all other proteins and no detectable inhibitory activity. Purified virus transduced NIH 3T3 murine fibroblasts 21 to 186-fold more efficiently than virus that was not purified. In addition, virus purification significantly altered the dose response of transduction. When virus that had not been purified was used to transduce cells, the relationship between transduction and virus concentration was highly non-linear. In contrast, when purified virus was used, transduction increased monotonically and was linearly proportional to virus concentration, except when high doses of virus were used. Interestingly, when high doses of virus were used gene transfer reached a maximum plateau level, most likely because particle-associated amphotropic envelope proteins had saturated the cellular receptors for the virus. Our findings illustrate that retrovirus purification increases the maximum number of genes that can be transferred, reduces the amount of virus required to achieve a given level of gene transfer, and reduces uncertainties about the relationship between the amount of virus used and the number of genes transferred.