Pilot-scale verification of a computer-based simulation for the centrifugal recovery of biological particles

The development of a model for simulating the recoveries by pilot-scale disc-stack centrifugation of whole yeast cells, yeast cell debris and protein precipitates prepared by ammonium sulphate salting-out is presented. The model is based on the grade efficiency concept and incorporates the effects of hindered settling at high biomass concentrations and the breakage of shear-sensitive material within the centrifuge feed-zone to give an accurate prediction of solid/liquid separation. The simulations have been proven by comparison with data from pilot-scale verification trials. The trials have highlighted where improvements to the models were required to increase their accuracy. The value of verification trials in proving the validity of models is commented upon.     

Improved fractional precipitation method for purification of paclitaxel

This study investigated changing the methanol/water ratio during fractional precipitation of paclitaxel, and adding all the distilled water at room temperature, followed mixing for an additional 10 min. When the methanol/water ratio was 50:50, 40:60, and 30:70 (v/v), the paclitaxel yield was 42.0%, 84.3%, and 92.0%, respectively. When using a methanol/water ratio of 50:50 (v/v), a similar high purity and yield of paclitaxel to the case of storing at a low temperature was achieved when adding all the distilled water at room temperature, followed by additional mixing for 10 min and further mixing at room temperature during fractional precipitation. Thus, additional mixing after adding all the distilled water is confirmed as important during fractional precipitation. Furthermore, the present results show that a high yield of high-purity paclitaxel is possible with additional mixing at room temperature after adding all the distilled water, which is significantly more economical than the existing method of storing at a low temperature for a long time after adding all the distilled water during fractional precipitation.     

Improvement of fractional precipitation process for pre-purification of paclitaxel

Fractional precipitation is a simple, efficient method for pre-purifying paclitaxel extracted from plant cell cultures. However, the fractional precipitation process has been inherently problematic due to the lengthy precipitation time (～3 days) that is required. An improved fractional precipitation process could significantly reduce the precipitation time by increasing the purity of crude extract and the surface area available for precipitation. Glass beads (7 mm) were used to increase the surface area, and the optimal surface area per working volume (i.e. volume of reaction solution) (S/V) for achieving the highest purity and yield of paclitaxel possible was found to be 0.428 mm^?1. The content of paclitaxel dissolved in methanol that can be processed during fractional precipitation was evaluated, and it was established that up to 0.9% (w/v) pure paclitaxel content could be processed. This improved pre-purification process serves to minimize solvent usage and the size and complexity of the high performance liquid chromatography operation required for paclitaxel purification.     

A protocol for computer-based protein structure and function prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.     

Development and optimization of fractional precipitation for the pre-purification of (+)-dihydromyricetin

A novel fractional precipitation process, both simple and efficient, was developed for producing (+)-dihydromyricetin in high purity and high yield from crude extracts. The optimal acetone composition in water, initial (+)-dihydromyricetin concentration in crude extract, storage temperature, storage time, and pH were 1/5 (v/v), 0.1 g/mL, 4°C, 32 h, and 9.0, respectively. Crude extracts were efficiently pre-purified using fractional precipitation of (+)-dihydromyricetin by differences of solubility in an acetone solution, increasing purity from 55.0 to over 84.9% with an overall yield of 97.5%. The use of fractional precipitation for pre-purification allowed for rapid and efficient separation of (+)-dihydromyricetin from interfering compounds and dramatically increased the purity of crude (+)-dihydromyricetin for subsequent purification steps.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH,a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin,is a promising therapeutic candidate for thromboembolic diseases.To develop SFH into a new thrombolytic agent,scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies.Here,we describe a pilot-scale cultivation and purification process for the production of SFH.A high-cell-density fed-batch cultivation for the production of SFH in E.coli was developed in a 40-L bioreactor,which produced about 1.1 g/L of recombinant protein.SFH was purified to homogeneity from the E.coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography,with over 99% purity and 54% recovery.Moreover,the residual endotoxin content was less than 0.5 EU/mL.The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay,respectively.     

Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.     

Evaluation of a high surface area fractional precipitation process for the purification of paclitaxel from Taxus chinensis

In this study, we evaluated a high surface area fractional precipitation process to achieve the effective purification of paclitaxel, an anticancer agent, from plant cell cultures. Fractional precipitation experiments were performed by increasing the surface area per working volume (S/V) to 0.428/mm using a variety of ion exchange resins. When the cation exchange resin Amberlite IR 120 H was used, the highest purity (>85%) and yield (>80%) of paclitaxel could be obtained in the shortest fractional precipitation time (within 6 h). Use of an ion exchange resin resulted in the production of smaller paclitaxel precipitates since it inhibited the growth of particles. When Amberlite IR 120 H in particular was used, paclitaxel particles were 2 ?? 3 times smaller (less than 30 ??m radius) than those obtained in the absence of ion exchange resin. Paclitaxel particle size was inversely correlated with the zeta potential of the fractional precipitation solution after the addition of ion exchange resin.     

Process characterization strategy for a precipitation step for host cell protein reduction

Process characterization using QbD approaches has rarely been described for precipitation steps used for impurity removal in biopharmaceutical processes. We propose a two-step approach for process characterization in which the first step focuses on product quality and the second focuses on process performance. This approach provides an efficient, streamlined strategy for the characterization of precipitation steps under the Quality by Design paradigm. This strategy is demonstrated by a case study for the characterization of a precipitation using sodium caprylate to reduce host cell proteins (HCP) during a monoclonal antibody purification process. Process parameters were methodically selected through a risk assessment based on prior development data and scientific knowledge described in the literature. The characterization studies used two multivariate blocks to decouple and distinguish the impact of product quality (e.g., measured HCP of the recovered product from the precipitation) and process performance (e.g., step yield). Robustness of the precipitation step was further demonstrated through linkage studies across the overall purification process. HCP levels could be robustly reduced to ≤100 ppm in the drug substance when the precipitation step operated within an operation space of ≤1% (m/v) sodium caprylate, pH 5.0–6.0, and filter flux ≤300 L/m²-hr for a load HCP concentration up to 19,000 ppm. This two-step approach for characterization of precipitation steps has several advantages, including tailoring of the experimental design and scale-down model to the intended purpose for each step, use of a manageable number of experiments without compromising scientific understanding, and limited time and material consumption.     

Pilot-scale purification of zeamatin, an antifungal protein from maize

Zeamatin is an antifungal protein isolated from the seeds of Zea mays. A practical process for isolation and purification was developed to increase recovery yields for future testing of zeamatin as a novel therapeutic drug. Zeamatin was extracted with buffer from corn meal milled to flour. Solids were removed from the extract using pressure filtration, and zeamatin was purified using two separate reverse-phase chromatography steps. Determination of the amino-terminal amino acid sequence, Western blotting using anti-zeamatin antibody, and activity against Candida albicans were used to confirm that the purified protein was zeamatin. From 5 kg of corn meal, approximately 110 mg of zeamatin was purified to apparent homogeneity.     

The fractional precipitation of soil humic acid by ammonium sulphate

Summary A number of fractions have been obtained from a soil humic acid by salting-out and by leaching with ammonium sulphate. The fractions obtained are characterised by infra-red and ultra-violet spectroscopy and osmometry. The fractions removed at relatively low saturation with ammonium sulphate are less highly charged and contain more aliphatic hydrocarbon groups per unit weight than the fractions precipitated at high concentrations of salt. In addition, the number average molecular weight of successive fractions precipitated with ammonium sulphate decreases significantly.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.     

Targeted mass spectrometry approaches for protein biomarker verification

The search for protein biomarkers has been a highly pursued topic in the proteomics community in the last decade. This relentless search is due to the constant need for validated biomarkers that could facilitate disease risk stratification, disease diagnosis, prognosis, monitoring as well as drug development, which ultimately would improve our quality of life. The recent development of proteomic technologies including the advancement of mass spectrometers with high sensitivity and speed has greatly advanced the discovery of potential biomarkers. One of the bottlenecks lies in the development of well-established verification assays to screen the biomarker candidates identified in the discovery stage. Recently, absolute quantitation using multiple-reaction monitoring mass spectrometry (MRM-MS) in combination with isotope-labeled internal standards has been extensively investigated as a tool for high-throughput protein biomarker verification. In this review, we describe and discuss recent developments and applications of MRM-MS methods for biomarker verification.     

Evaluation of feeding and mixing conditions for fractional precipitation of paclitaxel from plant cell cultures

Fractional precipitation based on the difference in solubility of crude paclitaxel dissolved in methanol, to which distilled water must be added, is the most effective method for the pre-purification of paclitaxel. In this study, the effect of the distilled water feeding and mixing method on the efficiency of fractional precipitation and the formation of paclitaxel precipitate were evaluated. When the distilled water was added all at once, the highest purity (∼57.0%) and yield (∼81.0%) were obtained, and a spherical precipitate was formed by the clustering of crystal branches. On the other hand, when the distilled water was added intermittently in several aliquots, the purity and yield tended to decrease with increasing number of additions, and the precipitate took the form of a cross or pentagon with less clustering of branches. Not mixing after the addition of distilled water resulted in high purity (∼57.0%) and the formation of a spherical precipitate that showed increased branching around the nucleus over time. In contrast, when the sample was mixed intermittently after adding distilled water, paclitaxel was obtained in high yield (∼99.7%). Continuously mixing the sample after adding distilled water, however, caused the precipitate crystals to be broken into smaller pieces.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

Solvent-dependent precipitation of prion protein

To mediate adaptation to stimuli, the methyltransferase (CheR) catalyzes methyl group transfer from S-adenosyl-L-methionine (SAM) to glutamyl residues in the transmembrane receptors of the bacterial chemosensory signaling pathway. The interaction between receptors and CheR occurs at two sites: a methylation site-active site interaction, and a 'docking' site interaction that is separated both from the methylation sites and the CheR active site. It is not certain if the docking site interaction functions merely to localize the transferase in close proximity to the methylation sites, or if it also increases CheR catalytic activity. Isothermal titration calorimetry experiments are conducted to test for allosteric interactions between the docking and active sites on CheR, which are expected to be present if docking activates CheR. The binding parameters (DeltaG, DeltaH, DeltaS) of a substrate analog of SAM, S-adenosyl-L-homocysteine (SAH), are measured both in the absence and presence of saturating concentrations of a pentapeptide (NWETF) that defines the docking receptor docking segment. SAH binding is unaffected by the presence of saturating NWETF, providing evidence that an allosteric activation of CheR does not take place upon docking, and thus supports the idea that the CheR-NWETF interaction merely functions to localize CheR near the sites of methylation.