HPLC Purification Step Research Articles

Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin. Herein, we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales, after a single HPLC purification step. Through these synthetic proteins, a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed, which would help to delineate the structure–function relationships in other sulfated proteins and more importantly, to serve as a basis for the development of related antithrombotic agents.

Purification of surfactin compounds produced by a Bacillus subtilis strain

Surfactins are lipopeptide-type biosurfactants produced mainly by Bacillus species containing a peptide loop of seven amino acids and a hydrophobic fatty acid chain. These molecules exhibit various biological activities; therefore, their therapeutic and environmental applications are in the focus worldwide. In our work, a multi-step purification and separation process was developed to isolate surfactins from the ferment broth of B. subtilis SZMC 6179J strain. The process incorporates normal phase flash chromatography for pre-purifying the crude extract and two consecutive reverse phase HPLC separations for the isolation of the various surfactin molecules. The determination of the relative amounts of lipopeptides both in the crude extract and in each fraction of every separation step were carried out by HPLC-HESI-MS examinations. The ratio of surfactins in the crude extract was 21.35%, but after the preparative flash chromatographic separation the relative amount of surfactins was observed to be 30.44%. The preparative HPLC purification step resulted 85.39% purity of the surfactins. Nine different surfactin variants were isolated and identified from the fractions of this final semi-preparative HPLC purification, out of which three compounds were completely purified, and three others were detected in relative amounts of more than 95% in some fractions.

New synthesis of 6″-[18 F]fluoromaltotriose for positron emission tomography imaging of bacterial infection.

6″-[18 F]fluoromaltotriose is a positron emission tomography tracer that can differentiate between bacterial infection and inflammation in vivo. Bacteria-specific uptake of 6″-[18 F]fluoromaltotriose is attributed to the targeting of maltodextrin transporter in bacteria that is absent in mammalian cells. Herein, we report a new synthesis of 6″-[18 F]fluoromaltotriose as a key step for its clinical translation. In comparison with the previously reported synthesis, the new synthesis features unambiguous assignment of the fluorine-18 position on the maltotriose unit. The new method utilizes direct fluorination of 2″,3″,4″-tri-O-acetyl-6″-O-trifyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose followed by basic hydrolysis. Radiolabeling of the new maltotriose triflate precursor proceeds using a single HPLC purification step, which results in shorter reaction time in comparison with the previously reported synthesis. Successful synthesis of 6″-[18 F]fluoromaltotriose has been achieved in 3.5 ± 0.3% radiochemical yield (decay corrected, n = 7) and radiochemical purity above 95%. The efficient radiosynthesis of 6″-[18 F]fluoromaltotriose would be critical in advancing this positron emission tomography tracer into clinical trials for imaging bacterial infections.

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC-C-IRMS.

Prednisone and prednisolone are two anti-inflammatory steroidal drugs listed by the World Anti-Doping Agency (WADA) within the class of glucocorticoids, which are prohibited "in competition" and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC-C-IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ13 C value reference range (-28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC-C-IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro-11-deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC-C-IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.

Automated SPE-based synthesis of 16α-[18F]fluoroestradiol without HPLC purification step

[18F]fluoroestradiol ([18F]FES) is well-established PET radiotracer for diagnosing and monitoring treatment of estrogen-positive breast cancer. The radiotracer is produced via one-pot two steps synthesis using cyclic sulfate precursor and is usually purified by semi-preparative HPLC. Here we suggested simple SPE purification procedure using OASIS WAX 3cc and Sep-Pak QMA light cartridges that afforded [18F]FES in typically 15% RCY (corrected for decay) within 45 min formulated in 5% EtOH/saline. All purity parameters were well within specifications recommended in the Investigator's Brochure for [18F]Fluoroestradiol.

Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls

The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity.Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure.Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.

Mast cell degranulation via MRGPRX2 by isolated human albumin fragments

BackgroundMast cells are important modulators of the human immune system via their release of several inflammatory mediators and proteases. The release can be activated by different pathways: the classical immunoglobulin E-dependent pathway and by the non-immunological immunoglobulin E-independent pathway. MAS-related G protein-coupled receptor X2 (MRGPRX2) is expressed in mast cells and it is one of the endogenous receptor responsible for the IgE-independent activation of human mast cell. The MRGPRX2 is classified as orphan receptor and unlike most GPCRs, the MRGPRX2 recognizes a wide range of basic molecules. Thus, there still might be several unknown ligands for the receptor. MethodsMRGPRX2 activating peptides were isolated from human plasma using consecutive HPLC purification steps. The isolation process was monitored with MRGPRX2 transfected HEK 293 cells. The isolated peptides were sequenced by MS and synthetized. The synthetic peptides were used to determine degranulation of the human LAD 2 mast cell line by measuring β-hexosaminidase release. ResultsThree endogenous MRGPRX2 activating peptides were isolated from human plasma. These peptides are identified as fragments of albumin. The isolated fragments activate MRGPRX2 and degranulate MRGPRX2 expressing LAD 2 cells in dose-dependent manner. ConclusionsThe isolated basic peptides generated from human albumin are able to degranulate mast cells via the MRGPRX2. General significanceThese endogenous albumin fragments, cleaved from albumin by mast cell secreted proteases, provide a possible pathway for self-perpetuating mast cell dependent inflammation.

Total chemical synthesis of proteins without HPLC purification.

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.

Using 5-deoxy-5-[18F]fluororibose to glycosylate peptides for positron emission tomography

So far seven peptide-based (18)F-radiopharmaceuticals for diagnostic applications with positron emission tomography (PET) have entered into clinical trials. Three candidates out of these seven are glycosylated peptides, which may be explained by the beneficial influence of glycosylation on in vivo pharmacokinetics of peptide tracers. This protocol describes the method for labeling peptides with 5-deoxy-5-[(18)F]fluororibose ([(18)F]FDR) as a prosthetic group. The synthesis of [(18)F]FDR is effected by a nucleophilic fluorination step by using dried Kryptofix 2.2.2-K2CO3-K(18)F complex and a subsequent HCl-catalyzed hydrolysis. The conjugation of [(18)F]FDR to the N-terminus aminooxy (-ONH2)-functionalized peptides is carried out in anilinium buffer at pH 4.6 and at room temperature (RT, 21-23 °C), with the concentration of peptide precursors being 0.3 mM. The procedure takes about 120 min and includes two cartridge isolation steps and two reversed-phase (RP) HPLC purification steps. The quaternary methyl amine (QMA) anion exchange cartridge and the hydrophilic-lipophilic balanced (HLB) cartridge are used for the isolation of (18)F-fluoride and [(18)F]FDR-conjugated peptides, respectively. The first HPLC purification provides the (18)F-fluorinated precursor of [(18)F]FDR and the second HPLC purification is to separate labeled peptides from their unlabeled precursors. The final product is formulated in PBS ready for injection, with a radiochemical purity of >98% and a radiochemical yield (RCY) of 27-37% starting from the end of bombardment (EOB). The carbohydrate nature of [(18)F]FDR and the operational convenience of this protocol should facilitate its general use.

GSP-37, a novel goldfish scale matrix protein: identification, localization and functional analysis

A novel noncollagenous acidic protein was identified from the scales of goldfish (Carassius auratus), a freshwater teleost. Using an in vitro calcium phosphate crystallization assay, the EDTA-soluble fraction from these scales was screened for crystallization inhibitory activity, and a highly phosphorylated glycoprotein, named goldfish scale protein (GSP)-37, was isolated through 5 HPLC purification steps. The cDNA for GSP-37 has an open reading frame encoding a precursor protein, consisting of a signal peptide and GSP-37, with 19 and 137 amino acid residues, respectively. The C-terminal region of GSP-37 contains the RGD consensus sequence for cell adhesion. Although native GSP-37 strongly inhibited crystallization, alkaline phosphatase treatment dramatically reduced its inhibitory activity. Reverse transcription-PCR analysis revealed that GSP-37 is expressed only in scales but not in other calcified tissues, bones or pharyngeal teeth. In situ hybridization demonstrated that GSP-37-expressing cells were localized in the central regions of regenerating scales, where organic matrices were actively synthesized and were not stained with either alkaline phosphatase or tartrate-resistant acidic phosphatase, osteoblastic and osteoclastic cell markers, respectively. Immunohistochemical analyses showed that GSP-37 is localized in the uppermost region of the bony layer of the scale, which is thought to correspond to the enamel or enameloid layer of vertebrate teeth. All these data strongly indicate that GSP-37 is deeply associated with calcification in fish scales.

Fully automated SPE-based synthesis and purification of 2-[18F]fluoroethyl-choline for human use

2-[(18)F]Fluoroethyl-choline ([(18)F]FECH) is a promising tracer for the detection of prostate cancer as well as brain tumors with positron emission tomography (PET). [(18)F]FECH is actively transported into mammalian cells, becomes phosphorylated by choline kinase and gets incorporated into the cell membrane after being metabolized to phosphatidylcholine. So far, its synthesis is a two-step procedure involving at least one HPLC purification step. To allow a wider dissemination of this tracer, finding a purification method avoiding HPLC is highly desirable and would result in easier accessibility and more reliable production of [(18)F]FECH. [(18)F]FECH was synthesized by reaction of 2-bromo-1-[(18)F]fluoroethane ([(18)F]BFE) with dimethylaminoethanol (DMAE) in DMSO. We applied a novel and very reliable work-up procedure for the synthesis of [(18)F]BFE. Based on a combination of three different solid-phase cartridges, the purification of [(18)F]BFE from its precursor 2-bromoethyl-4-nitrobenzenesulfonate (BENos) could be achieved without using HPLC. Following the subsequent reaction of the purified [(18)F]BFE with DMAE, the final product [(18)F]FECH was obtained as a sterile solution by passing the crude reaction mixture through a combination of two CM plus cartridges and a sterile filter. The fully automated synthesis was performed using as well a Raytest SynChrom module (Raytest, Germany) or a Scintomics HotboxIII module (Scintomics, Germany). The radiotracer [(18)F]FECH can be synthesized in reliable radiochemical yields (RCY) of 37±5% (Synchrom module) and 33±5% (Hotbox III unit) in less than 1 h using these two fully automated commercially available synthesis units without HPLC involvement for purification. Detailed quality control of the final injectable [(18)F]FECH solution proved the high radiochemical purity and the absence of Kryptofix2.2.2, DMAE and DMSO used in the course of synthesis. Sterility and bacterial endotoxin testing following standard procedures verified that the described production method for [(18)F]FECH is suitable for human applications. The routine production of [(18)F]FECH with sufficient RCYs was established by reliable and fast solid-phase extraction purifications of both the secondary labeling precursor [(18)F]BFE and the final product [(18)F]FECH, avoiding complex and sensitive HPLC equipment. The purity of the product was >95%, rendering the tracer suitable for human application. The newly developed purification procedure for [(18)F]BFE significantly reduces the complexity of the automated synthesis unit, hence reducing the cost for routine production in a clinical setup and allowing easy transfer to different synthesis modules.

A multi-technique approach using LC–NMR, LC–MS, semi-preparative HPLC, HR–NMR and HR–MS for the isolation and characterization of low-level unknown impurities in GW876008, a novel corticotropin-release factor 1 antagonist

A multi-technique approach was applied in order to fully characterize four low-level unknown impurities of GW876008, a novel CRF 1 receptor antagonist. Liquid chromatography (LC)–NMR spectroscopy was used in combination with LC–MS to obtain detailed information regarding the structure of the two major impurities present in batches of GW876008 and observed in the first synthetic scale-up for preclinical use. Two additional impurities were unexpectedly found at greater levels in a large scale synthesis for clinical use and their structure was elucidated by means of high resolution (HR)–MS and HR–NMR, after a small scale preparative HPLC purification step. This structural information was useful in terms of shedding light on the typical impurity profile of this new chemical entity with the aim to support the early development package for Phase I clinical studies.

A new “era” for cyclotide sequencing

In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Natures ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts ofOldenlandia affinis and Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinis extract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new "era" for cyclotide sequencing.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Comparison of two full automatic synthesis methods of 9-(4-[ 18F]fluoro-3-hydroxymethylbutyl)guanine using different chemistry modules

We have developed synthesis methods for 9-(4-[ 18F]fluoro-3-hydroxymethylbutyl)guanine ([ 18F]FHBG) using two commercial automatic chemistry modules, Tracerlab MX and Explora RN, and compared radiochemical yields. Synthesis conditions and sequence programs were modified for two modules because both these modules have different mechanical structures, including heater type, vacuum system, reactor, and tubing size. Synthesis using the Tracerlab MX module showed a 21.0±3.8% yield of radiochemical, which was 98±0.9% pure; the total preparation time was 63.0±5.0 min including an HPLC purification step. In contrast, synthesis using the Explora RN module showed a 32.0±1.2% yield of radiochemical, which was 99.0±0.6% pure; the total preparation time was 38±2 min, using different HPLC purification conditions and without the HPLC solvent evaporation step.

Development of an Enzyme-Linked Immunosorbent Assay for Serum 11-Dehydrocorticosterone in Rat and Mouse

We have developed an enzyme-linked immunosorbent assay (ELISA) for serum 11-dehydrocorticosterone (4-pregnen-21-ol-3,11,20-trione). The antiserum against 11-dehydrocorticosterone 21-hemisuccinate-conjugated bovine serum albumin was raised in rabbits. As an enzyme-labeled antigen, 11-dehydrocorticosterone 21-hemisuccinate was conjugated to horseradish peroxidase. Two ELISA systems were established: one without the extraction of steroids from serum (direct method), and another that used an HPLC purification step (HPLC method). The cross-reactivity of all steroids tested against the antibody was low except cortisone (92%); however, since cortisone levels in rats and mice are negligible, cortisone does not interfere with this direct ELISA. The measurable range of serum 11-dehydrocortiocosterone in both the direct and HPLC methods was 0.3-250 ng/ml and 0.78-400 ng/ml, respectively. Both methods displayed satisfactory parallel dilution, recovery and reproducibility; moreover, the values obtained with each method significantly correlated with the alternate method. To evaluate the two ELISA systems, the serum concentrations of 11-dehydrocorticosterone in normal rats and mice were determined by these two systems. The levels in Wistar rats fluctuated from 3 to 14 weeks of age (7.8+/-2.6 ng/ml) but at 1 week (1.7+/-1.2 ng/ml) were significantly low compared to other ages. No sex differences were found in rats and mice. Further, using the proposed direct method, chronological changes of rat serum 11-dehydrocorticosterone levels after 11-dehydrocorticosterone administration have been investigated together with corticosterone levels. These results verify that the proposed ELISA for 11-dehydrocorticosterone is useful for measuring 11beta-HSD activities in combination with the determination of serum corticosterone in rats and mice.

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.

BPMSS – Lonza's Biopharmaceutical Small Scale Plant Started cGMP Manufacturing in September 2004

In September 2004 Lonza (Visp, Switzerland) started operations in a new state of the art facility for cGMP manufacture of injectable grade microbially derived biopharmaceuticals. Both intracellular and secreted products can be processed in the new production line, which delivers bulk drug substances. cGMP conformity, including clean room classification, was cross-checked during the design phase with the Swiss regulatory authorities. The layout of the highly automated multi-product line is based on a high performance 1000 l bioreactor suitable for high cell density fermentation. Besides standard chromatography and ultrafiltration, the ex-proof design in upstream and downstream areas allows for solvent feeds in fermentation and for implementation of HPLC purification steps in protein and peptide purification.

Synthesis of a technetium‐99m labelled L‐tyrosine derivative with the fac‐99mTc(I)(CO)3‐core using a simple kit‐procedure

AbstractThe synthesis of a novel technetium‐99m labelled derivative of L‐tyrosine as a potential tumour imaging agent for nuclear medicine diagnosis is reported. The synthesis involved the labelling precursor fac‐[99mTc(OH2)(CO)3]+ which was synthesized using the commercially available Isolink®‐labelling kit and the tyrosine derivative O‐(N,N‐bis(carboxymethyl)aminoethyl)‐L‐tyrosine trifluoroacetate. The labelled compound O‐(99mTc(I)‐tricarbonyl‐N,N‐bis(carboxymethyl)aminoethyl)‐L‐tyrosine was obtained in a radiochemical yield of 70–80% within 60 min with a radiochemical purity greater than 98% without any HPLC purification step. Purification was achieved merely by solid phase extraction. Chemical as well as chiral purity was determined using gradient‐ and chiral HPLC. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Tandem IMAC–HPLC purification of a cocaine-binding scFv antibody

Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression. However, the high purity demands of certain applications are occasionally unattainable through a single IMAC separation. GNC92H2scFv is a cocaine-binding single-chain antibody fragment that is unstable during long-term storage in aqueous solution. To circumvent this problem, a reversed-phase HPLC separation was performed following IMAC purification of GNC92H2scFv from Escherichia coli cell culture supernatant. The resulting HPLC effluent was then freeze-dried to afford a salt-free lyophilizate amenable to long-term storage with minimal loss in binding activity. HPLC purification also effectively removed an 80-kDa protein contaminant that co-eluted with the IMAC-purified protein. Of special importance for in vivo applications of recombinantly expressed protein therapeutics, an HPLC purification step afforded a 1000-fold reduction in lipopolysaccharide (LPS) endotoxin contamination in the final GNC92H2scFv product.