共查询到20条相似文献,搜索用时 15 毫秒
1.
Anton Löfgren Mikael Yamanee-Nolin Simon Tallvod Joaquín G. Fons Niklas Andersson Bernt Nilsson 《Biotechnology progress》2019,35(6):e2871
With continued development of integrated and continuous downstream purification processes, tuning and optimization become increasingly complicated with additional parameters and codependent variables over the sequence. This article offers a novel perspective of nonlinear optimization of integrated sequences with regard to individual column sizes, flow rates, and scheduling. The problem setup itself is a versatile tool to be used in downstream design which is demonstrated in two case studies: a four-column integrated sequence and a continuously loaded twin-capture setup with five columns. 相似文献
2.
3.
Evaluation of several protein a resins for application to multicolumn chromatography for the rapid purification of fed‐batch bioreactors
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nicolas‐Julian Hilbold Xavier Le Saoût Eric Valery Laurence Muhr Jonathan Souquet Alain Lamproye Hervé Broly 《Biotechnology progress》2017,33(4):941-953
Most of the existing production capacity is based on fed‐batch bioreactors. Thanks to the development of more efficient cell lines and the development of high‐performance culture media, cell productivity dramatically increased. In a manufacturing perspective, it is necessary to clear as quickly as possible the protein A capture step to respect the manufacturing agenda. This article describes the methodology applied for the design of a multicolumn chromatography process with the objective of purifying as quickly as possible 1,000 and 15,000 L fed‐batch bioreactors. Several recent and reference protein A resins are compared based on characteristic values obtained from breakthrough curves. The importance and relevance of resin parameters are explained, and purposely simple indicators are proposed to quickly evaluate the potential of each candidate. Based on simulation data, the optimum BioSC systems associated with each resin are then compared. The quality of the elution delivered by each resin is also compared to complete the assessment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:941–953, 2017 相似文献
4.
Automated genome sequence analysis and annotation. 总被引:5,自引:0,他引:5
M A Andrade N P Brown C Leroy S Hoersch A de Daruvar C Reich A Franchini J Tamames A Valencia C Ouzounis C Sander 《Bioinformatics (Oxford, England)》1999,15(5):391-412
MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit 相似文献
5.
Randolph V. Lewis Michael Hinman Srinivas Kothakota Maurille J. Fournier 《Protein expression and purification》1996,7(4):400-406
Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced inEscherichia coliat levels up to 10 mg/g wet wt of cells although yields of 1–2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers. 相似文献
6.
Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nikhil Kateja Harshit Agarwal Vishwanath Hebbi Anurag S. Rathore 《Biotechnology progress》2017,33(4):998-1009
Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end‐to‐end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three‐column periodic counter‐current chromatography for capture of the product followed by a three‐column con‐current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end‐to‐end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998–1009, 2017 相似文献
7.
The effect of column verticality on separation efficiency in expanded bed adsorption 总被引:1,自引:0,他引:1
The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15° resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15°. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185° off-vertical. The G6PDH breakthrough curves for vertical and 0.15° off-vertical runs performed using a 5 cm column were essentially indistinguishable. 相似文献
8.
Gregory A. Norris 《The International Journal of Life Cycle Assessment》2001,6(2):118-120
The private sector decision making situations which LCA addresses mustalso eventually take theeconomic consequences of alternative products or product designs into account. However, neither the internal nor external economic
aspects of the decisions are within the scope of developed LCA methodology, nor are they properly addressed by existing LCA
tools. This traditional separation of life cycle environmental assessment from economic analysis has limited the influence
and relevance of LCA for decision-making, and left uncharacterized the important relationships and trade-offs between the
economic and life cycle environmental performance of alternative product design decision scenarios. Still standard methods
of LCA can and have been tightly, logically, and practically integrated with standard methods for cost accounting, life cycle
cost analysis, and scenario-based economic risk modeling. The result is an ability to take both economic and environmental
performance — and their tradeoff relationships — into account in product/process design decision making. 相似文献
9.
Process development for biologics is expensive and lengthy, tools are needed to rapidly understand where the difficulties will lie, and, hence, rationally deploy resources. In this work we introduce and evaluate a methodology to determine the manufacturability of a protein candidate. The methodology determines protein impurities by mass spectrometry and separation difficulty from the product based on adsorption properties deduced from a single set of experiments. This information can aid early process strategy decisions to target hard to remove protein impurities (nearest neighbors) and allow the re-evaluation of conventional process synthesis. The methodology chosen gives consideration to the fact that at this point in early phase development, material, and established analytical methods are limiting. This study uses surface enhanced laser desorption ionization mass spectroscopy (SELDI-MS), for its rapid analysis and minimal sample requirement to measure product and contaminant adsorption properties. The technique is used to provide an array of hydrophobic and electrostatic conditions for protein adsorption. The adsorption pattern produced for each protein is analyzed and visualized via a star plot. Dendrograms then define nearest neighbor protein contaminants by quantifying differences in the adsorption pattern between the product and contaminants. By comparison to an existing process to manufacture a 28 kDa recombinant protein expressed in Escherichia coli, we confirm the method is capable of determining where the greatest separation difficulty lies and what separation methods should be considered. The technique identified that the nearest neighbor contaminants were product-related proteins (28.6 and 29.1 kDa/e). Thus demonstrating a capability to measure the relative difficulty of purifying early stage protein candidates where little is known about the separation properties of products and contaminants, or the process sequence for their production. 相似文献
10.
Canarelli S Fisch I Freitag R 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,775(1):27-35
A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45% of the light and 75% of the heavy chain sequences of the antibody could be verified within minutes. 相似文献
11.
Tokunaga H Hara S Arakawa T Ishibashi M Gupta RS Tokunaga M 《Journal of Protein Chemistry》1999,18(8):837-844
The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH2-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column. 相似文献
12.
ProteoMix is a suite of JAVA programs for identifying, annotating and predicting regions of interest in large sets of amino acid sequences, according to systematic and consistent criteria. It is based on two concepts (1) the integration of results from different sequence analysis tools increases the prediction reliability; and (2) the integration protocol is critical and needs to be easily adaptable in a case-by-case manner. ProteoMix was designed to analyze simultaneously multiple protein sequences using several bioinformatics tools, merge the results of the analyses using logical functions and display them on an integrated viewer. In addition, new sequences can be added seamlessly to an analysis performed on an initial set of sequences. ProteoMix has a modular design, and bioinformatics tools are run on remote servers accessed using the Internet Simple Object Access Protocol (SOAP), ensuring the swift implementation of additional tools. ProteoMix has a user-friendly interactive graphical user interface environment and runs on PCs with Microsoft OS. AVAILABILITY: ProteoMix is freely available for academic users at http://bio.gsc.riken.jp/ProteoMix/ 相似文献
13.
Zoltán Pataj István Ilisz Róbert Berkecz Enikő Forró Ferenc Fülöp Antal Péter 《Chirality》2010,22(1):120-128
High‐performance liquid chromatographic methods were developed for the separation of the enantiomers of 19 β‐lactams. The direct separations were performed on chiral stationary phases containing either amylose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® AmyCoat? column) or cellulose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® CelluCoat? column) as chiral selector. The different methods were compared in systematic chromatographic examinations. The separations were carried out with good selectivity and resolution. The AmyCoat? and CelluCoat? columns appear to be highly complementary. The best separations of bi‐ and tricyclic β‐lactam stereoisomers were obtained with the AmyCoat? column, whereas the 4‐aryl‐substituted β‐lactams were better separated on the CelluCoat? column. The elution sequence was determined in all cases; no general rule could be established. Chirality 2010. © 2009 Wiley‐Liss, Inc. 相似文献
14.
Larson SM England JL Desjarlais JR Pande VS 《Protein science : a publication of the Protein Society》2002,11(12):2804-2813
Modeling the inherent flexibility of the protein backbone as part of computational protein design is necessary to capture the behavior of real proteins and is a prerequisite for the accurate exploration of protein sequence space. We present the results of a broad exploration of sequence space, with backbone flexibility, through a novel approach: large-scale protein design to structural ensembles. A distributed computing architecture has allowed us to generate hundreds of thousands of diverse sequences for a set of 253 naturally occurring proteins, allowing exciting insights into the nature of protein sequence space. Designing to a structural ensemble produces a much greater diversity of sequences than previous studies have reported, and homology searches using profiles derived from the designed sequences against the Protein Data Bank show that the relevance and quality of the sequences is not diminished. The designed sequences have greater overall diversity than corresponding natural sequence alignments, and no direct correlations are seen between the diversity of natural sequence alignments and the diversity of the corresponding designed sequences. For structures in the same fold, the sequence entropies of the designed sequences cluster together tightly. This tight clustering of sequence entropies within a fold and the separation of sequence entropy distributions for different folds suggest that the diversity of designed sequences is primarily determined by a structure's overall fold, and that the designability principle postulated from studies of simple models holds in real proteins. This has important implications for experimental protein design and engineering, as well as providing insight into protein evolution. 相似文献
15.
Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes. 相似文献
16.
Young G. Park 《Biotechnology and Bioprocess Engineering》2000,5(5):332-339
Experimental and theoretical works were performed for the separation of large polyelectrolytes such as DNA in the column packed
with gel particles under an electric field. This paper shows how intraparticle convection effects the separation of DNAs in
the column because DNAs quickly oriented through the pores in the field direction. Dimensionless transient mass balance equations
were derived considering diffusion and electrophoretic convection. The separation criteria is theoretically studied using
two different Peclet numbers in the fluid and solid phases and these criteria were verified uing two different DNAs by electrophoretic
mobilities measured experimentally, showing how the separation position of DNAs varies in the column according to values ofPe
f/Peg of individual DNA. Governing equations are simultaneously solved by operator theoretic and characteristic methods to yield
the column response. 相似文献
17.
Expression,purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli 总被引:1,自引:0,他引:1
Subramanian S Kondaiah P Radhakantha Adiga P 《Protein expression and purification》2002,26(2):284-289
Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals. 相似文献
18.
MOTIVATION: Protein sequence and family data is accumulating at such a rapid rate that state-of-the-art databases and interface tools are required to aid curators with their classifications. We have designed such a system, MetaFam, to facilitate the comparison and integration of public protein sequence and family data. This paper presents the global schema, integration issues, and query capabilities of MetaFam. RESULTS: MetaFam is an integrated data warehouse of information about protein families and their sequences. This data has been collected into a consistent global schema, and stored in an Oracle relational database. The warehouse implementation allows for quick removal of outdated data sets. In addition to the relational implementation of the primary schema, we have developed several derived tables that enable efficient access from data visualization and exploration tools. Through a series of straightforward SQL queries, we demonstrate the usefulness of this data warehouse for comparing protein family classifications and for functional assignment of new sequences. 相似文献
19.
This article documents the whole genome sequence information of the Indian Zaprionus indianus, a member of the fruit fly family Drosophilidae. The sequences were generated on an Illumina platform and reads and whole genome sequence submitted to NCBI to the SRA and BioProject databases, respectively. This is the first Indian Z. indianus whole genome (draft) submitted to the sequence repository with SRA reads. The details of methodology, assembly statistics and functional annotation are presented in this work. 相似文献
20.
A method based on Web-based tools is presented to design optimally functioning molecular beacons. Molecular beacons, fluorogenic hybridization probes, are a powerful tool for the rapid and specific detection of a particular nucleic acid sequence. However, their synthesis costs can be considerable. Since molecular beacon performance is based on its sequence, it is imperative to rationally design an optimal sequence before synthesis. The algorithm presented here uses simple Microsoft Excel formulas and macros to rank candidate sequences. This analysis is carried out using mfold structural predictions along with other free Web-based tools. For smaller laboratories where molecular beacons are not the focus of research, the public domain algorithm described here may be usefully employed to aid in molecular beacon design. 相似文献