A case study: oligonucleotide purification from gram to hundred gram scale

This article describes the purification and scale-up of ISIS 2302, a 20-mer phosphorothioate oligonucleotide by anion-exchange (AX) chromatography. The key operating parameters were optimized at gram scale and further scaled up to hundred gram. SOURCE 30Q, a high efficiency polymeric chromatographic media was used for both the small and large-scale work. High length-based purity and yield were maintained at scale-up. This purification is one of the largest demonstrations of AX purification of phosphorothioate oligonucleotide.     

Purification of a synthetic oligonucleotide by anion exchange chromatography: method optimisation and scale-up

A single-step chromatographic method for purification of a synthetic 20-mer oligonucleotide is described. Method optimisation was conducted at laboratory scale where 30 mg crude sample was purified per run with a yield of 17 mg pure oligonucleotide. The protocol was scaled-up in steps to achieve 5-, 58- and a final 230-fold scale-up. At the final scale, 7.0 g of crude material was purified with a yield of 4.1 g product. The purity of the oligonucleotide was in all scales higher than 97%. The cycle time was 110 min, which corresponds to a purification capacity of about 90 g crude oligonucleotide material per 24 h.     

Large-scale purification of oligonucleotides by extraction and precipitation with butanole

Today the synthesis of oligonucleotides is a well-established process. Using automatic synthesizers even kilogram quantities can be produced in a few hours. However, the purification of the final product is still time-consuming and needs a complex apparatus. In this article, a simple and fast purification method for the large-scale syntheses of oligonucleotides is described. According to the method of Sawadago and van Dyke ([1991] Nucleic Acids Res 19:674-675) for small-scale oligonucleotide purification, oligonucleotides in mumol to mmol amounts were purified by liquid-liquid extraction using butanole as the extraction liquid. Choosing appropriate ratios of extraction liquid to oligonucleotide solution, simultaneous purification and precipitation could be achieved. It was found that the yield of the purified oligonucleotide was mainly affected by the temperature. Yield decreased with increasing temperature. The use of this improved extraction procedure allows the purification of gram to kilogram quantities of oligonucleotides in less than a day with simple equipment and high yield.     

Some properties and applications of Superose 6B

Applications of a new agarose-based medium for high-performance preparative gel filtration is described. The 30-micron bead size, high pore volume, and simple experimental technique to prepare highly efficient columns make Superose 6B very suitable for preparative purifications of protein mixtures in the molar mass range of 10(3) to 4 X 10(6). Sample load exceeds 100 mg/cm2, which indicates that gram quantities may be purified on K 50/60 columns. The scale-up procedure from analytical to preparative columns is demonstrated by the separation of serum samples and the industrial purification of an enzyme-antibody conjugate.     

临床分离的革兰阴性细菌的耐药谱及耐药机制的研究

目的 了解前临床上分离G^－细菌的药敏状况和耐药机制及提供合理使用抗生素的依据。方法 主要使用MICROSCAN WALKAWAY／－40全自动微生物分析仪对1999年3月－2000年3月全院住院病人的尿、痰、腹水、脓液、创面、前列腺液、血液等培养呈阳性的标本进行细菌鉴定和药敏试验,结果共检出G^-菌1152株包括27个菌属80个菌种,觉细菌是大肠埃希菌（16.1％）、铜绿假单胞菌（6.5％）、肺炎克雷伯菌（5.3％）等。G^-杆菌（除不动杆菌外）对第三代头孢霉素敏感率已降到（3.0%-76.1%)、对亚胺培南（80.7%-92%)、头孢哌酮／舒巴坦（58.8％－100％）、阿米卡星（41.4%-93.2%)、环丙沙星（30.5％－67.3％）较敏感;对第三代头孢霉素产生超广谱β－内酰胺酶（ESBLs）,肺炎克雷伯菌高达35.0%-36.9%,大肠5埃希菌达21.8％－23％。对常用β-内酰胺类抗生素产诱导酶（IB）,铜绿假单胸菌高达51％－60.9％,弗劳地枸橼酸菌达4.5％－63.6％,阴沟肠杆菌达8.7%-35.3％。结论 目前G^－杆菌对β-内酰胺类抗生素药的主要机制是产生ESBLs和IB0G^-杆菌引起的感染首选亚胺培南单用或第三代浆孢霉素复合制剂（头孢哌酮／舒巴坦）联合阿米卡星或氟喹酮类,第三代头孢霉素除非药敏提示否则不宜选用。     

中试规模纯化海洋芽孢杆菌源脂肽类化合物

本次研究旨在建立经济可行的海洋芽孢杆菌源脂肽类化合物的中试规模纯化工艺。对包括酸化沉淀、甲醇浸提、溶剂沉淀、盐析、萃取、硅胶柱层析和HZ806大孔树脂吸附工艺在内的可放大的成熟单元工艺进行反复试验,考察脂肽类化合物表面活性对单元工艺的影响。严格遵循以高收率为前提循序渐进逐步减少杂质的原则,组合上述单元工艺对目标产物进行提取和纯化,并最终获得高纯度脂肽样品。新工艺可从1 t海洋芽孢杆菌Bacillus marinus B-9987的发酵液中,以百克量级的规模制备87.51%–100%纯度的脂肽类化合物样品,收率81.73%。本研究首次实现了高纯度的海洋芽孢杆菌源脂肽类化合物的百克量级制备;允许发酵生产阶段使用天然培养基,缓解了脂肽中游发酵生产和下游大规模纯化之间的矛盾;且各单元工艺规避了脂肽类化合物水溶液的乳化起泡和不经济的大体积水溶液蒸发浓缩。新工艺实用可行,经济合理。     

Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.     

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

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

Large-scale purification of monoclonal antibodies

This article reviews aspects of the purification of monoclonal antibodies which are important for scale-up, and assesses the current status of large-scale processing of monoclonal antibodies.     

Novel, rapid purification of the membrane protein photosystem I by high-performance liquid chromatography on porous materials

New porous materials have been tested for their potential to speed up purification of membrane proteins. As an example the purification of photosystem I, a light-driven electron pump from the cyanobacterium Synechocystis PCC6803, was optimized. The combination of two HPLC steps (an anion-exchange chromatography followed by a hydrophobic interaction chromatography) yields homogeneous monomeric or trimeric photosystem I as determined by gel filtration and gel electrophoresis. In comparison to traditional purification schemes our method is at least three-times faster and allows for easy scale-up.     

Whole-blood immunoassay for γH2AX as a radiation biodosimetry assay with minimal sample preparation

The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0–5 Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.     

Pharmacokinetic and toxicity profile of a phosphorothioate oligonucleotide following inhalation delivery to lung in mice

Antisense oligonucleotides are currently being investigated for the treatment of a variety of diseases. Antisense drugs are being administered primarily by parenteral injection. To explore more convenient patient delivery methods, we have characterized the tissue kinetics and tolerability of an inhaled aerosol formulation of a phosphorothioate oligonucleotide in mice. Concentrations of oligonucleotide in bronchioalveolar lavage fluid, plasma, and tissue and immunohistochemical localization were used to assess deposition and pharmacokinetic parameters. Significant concentrations of oligonucleotide in lung, as well as systemic tissues, were measured following a pulmonary dose of 12 mg/kg. Doses as low as 1-3 mg/kg also produced significant concentrations of oligonucleotide (>50 microg oligonucleotide per gram of tissue), and these were maintained in the lung with a halflife of 20 hours or greater. Oligonucleotide was localized to bronchiolar epithelium and alveolar epithelium and endothelium. Toxicity was mild at the 12 mg/kg level and minimal to absent at doses of 3 mg/kg or below. Based on a favorable pharmacokinetic profile and a relative lack of toxicity, inhalation delivery appears to be a therapeutic option for antisense oligonucleotides.     

连续流层析技术在亲和层析中的应用及生产放大评估

生物制药行业迅速发展,尤其是上游表达量的增加和规模的扩大,促使上游培养采用连续灌流方式,同时也推动了下游纯化生产工艺相应的采取连续纯化策略.以灌流培养的Fc融合蛋白为例,采用BioSMB PD设备,对比了下游工艺亲和层析捕获步骤中单柱批次纯化和连续流层析纯化的样品纯度和收率,并在此基础上进行小试工艺放大和生产实际用量成...     

Purify First: rapid expression and purification of proteins from XMRV

Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Large-scale purification of recombinant human angiostatin

A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed.     

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.     

Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column

A method using a strong anion-exchange liquid-chromatography column, Mono-Q, has been developed for high-resolution analysis and purification of oligonucleotide dithioates, which were synthesized by an automated, solid-phase, phosphorothioamidite chemistry. High-resolution separation of oligonucleotide phosphorodithioates from monothiophosphate impurities was obtained. High-resolution separation was also demonstrated at pH 8. The separation of oligonucleotide dithioates was found to be linearly dependent on the number of sulfurs for the same sequence length. Thiocyanate, SCN-, as eluting anion, can be used to purify oligonucleotides containing a high percentage of phosphorodithioate linkages in lower salt concentrations and provides better separation than chloride as eluting anion.     

Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Radiation-induced H2AX phosphorylation and neural precursor apoptosis in the developing brain of mice

We showed that gamma irradiation of the developing mouse brain with 2 Gy induced a massive apoptosis of neural precursors but not of neurons within 24 h. Successive phosphorylation and dephosphorylation of histone H2AX have been linked to DNA breaks and repair. Similar numbers of nuclear foci of phosphorylated H2AX (gamma-H2AX) were found 1 h postirradiation in neural precursors and in neurons, suggesting that differences in radiosensitivity were not related to variations in the numbers of DNA double-strand breaks induced by radiation. Surviving neural precursors like neurons totally lost gamma-H2AX within 24 h after irradiation, but they had a slower kinetics of loss of gamma-H2AX foci. This suggests that the DNA repair machinery processed damage more slowly in these neural precursors in relation to their greater radiosensitivity. We also found a bright and diffuse gamma-H2AX staining of nuclei of cells at an early stage of apoptosis, whereas cells at later stages of apoptosis were unstained. This was probably related to phosphorylation and subsequent degradation of H2AX in the course of DNA fragmentation during apoptosis. Detection of gamma-H2AX-bright nuclei may thus be a useful marker of neural cells at an early stage of apoptosis.