Parametric study of a 6-column countercurrent solvent gradient purification (MCSGP) unit

The novel "multicolumn countercurrent solvent gradient purification" (MCSGP) process has been modeled for the purification of a polypeptide mixture characterized by a strong non-linear competitive adsorption isotherm. As a model system, the purification of an industrial polypeptide mixture containing 46% of the hormone calcitonin has been selected. The many impurities contained in the mixture have been lumped into three key impurities, which are selected as the ones eluting closer to the main component. The simulation model allows for a better understanding of the complex operating behavior of the multicolumn system, which has been experimentally investigated in a previous work. Through a systematic parametric analyses of the model behavior, the main operating parameters controlling the process performance in terms of purity and yield are investigated. The study of internal liquid and adsorbed phase concentration profiles along the unit for the different operating conditions allow elucidating the working principle of the new separation process. It is found that the MCSGP unit achieves much higher yields for a given product purity than the corresponding single-column batch units.     

Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP)

Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two‐step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP‐captured material in batch mode. Using multi‐modal anion exchange in bind‐elute mode, mAb was produced consistently within the purity specification. The benchmark was a state‐of‐the‐art 3‐step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2‐step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase. Biotechnol. Bioeng. 2010;107: 974–984. © 2010 Wiley Periodicals, Inc.     

Design and operation of a continuous integrated monoclonal antibody production process

The realization of an end‐to‐end integrated continuous lab‐scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter‐based cell‐retention, a continuous two column capture process, a virus inactivation step, a semi‐continuous polishing step (twin‐column MCSGP), and a batch‐wise flow‐through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity‐yield trade‐off of classical batch‐wise bind‐elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight‐through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end‐to‐end integration. The steady‐state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303–1313, 2017     

Translocation mechanism of long sugar chains across the maltoporin membrane channel

Maltoporin allows permeation of long maltodextrin chains. It tightly binds the amphiphilic sugar, offering both hydrophobic interactions with a helical lane of aromatic residues and H bonds with ionic side chains. The minimum-energy path of maltohexaose translocation is obtained by the conjugate peak refinement method, which optimizes a continuous string of conformers without applying constraints. This reveals that the protein is passive while the sugar glides screw-like along the aromatic lane. Near instant switching of sugar hydroxyl H bond partners results in two small energy barriers (of approximately 4 kcal/mol each) during register shift by one glucosyl unit, in agreement with a kinetic analysis of experimental dissociation rates for varying sugar chain lengths. Thus, maltoporin functions like an efficient translocation "enzyme," and the slow rate of the register shift (approximately 1/ms) is due to high collisional friction.     

Experimental Evaluation of the Impact of Intrinsic Process Parameters on the Performance of a Continuous Chromatographic Polishing Unit (MCSGP)

The semicontinuous twin‐column multicolumn countercurrent solvent gradient purification (MCSGP) process improves the trade‐off between purity and yield encountered in traditional batch chromatography, while its complexity, in terms of hardware requirements and process design, is reduced in comparison to process variants using more columns. In this study, the MCSGP process is experimentally characterized, specifically with respect to its unique degrees of freedom, i.e., the four switching times, which alternate the columns between interconnected and batch states. By means of isolation of the main charge isoform of an antibody, it is shown that purity is determined by the selection of the product collection window with negligible influence from the recycle phases. In addition, the amount of weak and strong impurities can be specifically attributed to the start and end of the collection, respectively. Due to higher abundance of weakly adsorbing impurities, the start of product collection influences productivity and yield more than the other switching times. Furthermore, most of the encountered tendencies scale between different loadings. The found trends can be rationalized from the corresponding batch chromatogram and therefore used during process design to obtain desirable process performances without extensive trial‐and‐error experimentation or complete model development and calibration.     

Process for Continuous Fab Production by Digestion of IgG

Intensified processing and end‐to‐end integrated continuous manufacturing are increasingly being considered in bioprocessing as an alternative to the current batch‐based technologies. Similar approaches can also be used at later stages of the production chain, such as in the post‐translational modifications that are often considered for therapeutic proteins. In this work, a process to intensify the enzymatic digestion of immunoglobulin G (IgG) and the purification of the resulting Fab fragment is developed. The process consists of the integration of a continuous packed‐bed reactor into a multicolumn chromatographic process. The integration is realized through the development of a novel multicolumn countercurrent solvent gradient purification (MCSGP) process, which, by adding a third column to the classical two‐column MCSGP process, allows for continuous loading and then straight‐through processing of the mixture leaving the reactor.     

Comparison of multi-column chromatography configurations through model-based optimization

Integrated continuous bioprocessing has been identified as the next important phase of evolution in biopharmaceutical manufacturing. Multiple platform technologies to enable continuous processing are being developed. Multi-column counter-current chromatography is a step in this direction to provide increased productivity and capacity utilization to capture biomolecules like monoclonal antibodies (mAbs) present in the reactor harvest and remove impurities. Model-based optimization of two prevalent multi-column designs, 3-column and 4-column periodic counter-current chromatography (PCC) was carried out for different concentrations of mAbs in the feed, durations of cleaning-in-place and equilibration protocols. The multi-objective optimization problem comprising three performance measures, namely, product yield, productivity, and capacity utilization was solved using the Radial basis function optimization technique. The superficial velocities during load, wash, and elute operations, along with durations of distinct stages present in the multi-column operations were considered as decision variables. Optimization results without the constraint on number of wash volumes showed that 3-Column PCC performs better than 4-Column PCC. For example, at a feed concentration of 1.2 mg/mL, productivity, yield and capacity utilization, respectively, were 0.024 mg/mL.s, 0.94, and 0.94 for 3-Column PCC and 0.017 mg/mL.s, 0.87, and 0.83 for 4-column PCC. Similar trends were observed at higher feed concentrations also. However, when the constraint on number of wash volumes is included, 4-Column PCC was found to result in consistent productivity and product yield under different operating conditions but at the expense of reduced capacity utilization.     

The Biological Records Centre–a data centre

The Biological Records Centre collects data on the occurrence of species at a particular time in a particular place. These data are used for the preparation of distribution maps, for lists of species from localities, and lists of localities for species.
The unit of recording at the national level is the 10 km square: from each square a list as complete as possible is collected. For common species presence alone is sufficient, but for rare or critical species more detailed data on locality and source are required.
Mapping data are stored on magnetic tape from which 80-column cards are generated by computer. These cards prepare distribution maps on an electric typewriter through a card reader. Details of locality, habitat, etc. are stored on two sets of 80-column individual record cards, one stored by species and the other by localities.
The Centre is encouraging the collection of data in counties, where the 2times2 km square is used, and for Europe where the 50 times 50 km square is proposed. It is important to establish not only biological records centres but a complete biological recording network.     

DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae

The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB''s requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.Vibrio cholerae, the etiologic agent of cholera, is a gram-negative bacterium which hosts several specific filamentous phages (1, 7, 8, 9, 10, 11, 13). CTXφ phage has been the most studied due to its role in pathogenicity and horizontal gene transfer (6). This phage is usually integrated into the genomes of toxigenic strains of V. cholerae, but it is also able to replicate directly from the bacterial chromosome (6) and to produce infective phage particles with potential for transducing the cholera toxin genes into nonpathogenic environmental strains (6, 13). Another filamentous phage important for its role in horizontal gene transfer is VGJφ, which is able to recombine with the CTXφ genome to originate a hybrid phage endowed with the full potential for virulence conversion. The hybrid phage shows an increased infectivity due to its specificity for the receptor mannose-sensitive hemagglutinin (receptor mannose-sensitive hemagglutinin pilus), which is ubiquitous among environmental strains (1, 2). Therefore, elucidating the biology of these phages is crucial for understanding the evolution of bacterial pathogenesis.The genomes of CTXφ and VGJφ carry the putative homologous rstB and open reading frame 112 (orf112) genes, respectively. The requirement of rstB for the integration of CTXφ into the bacterial chromosome has been described (14). However, the biochemical function of the gene product has not been elucidated. Genes rstB and orf112 are positional and size homologues of genes encoding single-stranded DNA (ssDNA)-binding proteins (SSB) in other filamentous phages (1). It is expected that the proteins encoded by rstB and orf112 exert similar functions in the biology of their respective phages (1). Thus, we wanted to evaluate the ssDNA-binding activity of these ORF products.To asses whether the Orf112 product and RstB have SSB activity, sufficient amounts of pure proteins are required. This paper describes quick purification protocols used to obtain both protein species and the evaluation of their DNA binding activities. The Orf112 protein was obtained from V. cholerae strain 569B (serogroup O1, Inaba classical biotype) infected with VGJφ, which expresses high levels of the protein. The infected bacteria were inoculated into 300 ml of LB broth and were cultured with shaking overnight at 200 rpm and 37°C. Parallel uninfected batches of 569B were also processed. Cells were collected by centrifugation for 15 min at 9,000 × g and at 4°C and stored at −20°C until processed.A recombinant rstB gene with a hexahistidine tag coding region fused to the C terminus of the respective protein product (RstB-His) was constructed by cloning the gene into the expression vector pBAD/Myc-HisC (Invitrogen). The rstB gene was PCR amplified from the CTXφ genome using the oligonucleotides CNC06-171 (5′-AGTTCCATGGGGAAATTATGGGTGATAAT-3′) and CNC06-173 (5′-CATCAAGCTTTAATGGGT-3′), which introduce restriction sites for NcoI and HindIII at the amplicon ends. The amplified fragment was digested with both enzymes and cloned into the same sites of pBAD/Myc-HisC. In the resultant construction, named pBAD/Myc-HisC-rstB 9, expression of the recombinant protein is inducible by arabinose.Plasmid pBAD/Myc-HisC-rstB 9 was electroporated into Escherichia coli Top 10. A 1-ml sample of an overnight, 5-ml, ampicillin-supplemented LB broth culture of transformed Top 10 was inoculated into 300 ml of fresh broth. The culture was incubated with orbital shaking at 200 rpm and 37°C until it reached an optical density at 600 nm of 0.5. To induce expression of the RstB-His protein, 0.002% (wt/vol) arabinose was added and the culture was reincubated for three additional hours. Parallel batches of pBAD/Myc-HisC-transformed E. coli Top 10 were processed as a negative control. Cells were sedimented by centrifugation for 15 min at 9,000 × g and 4°C and stored at −20°C until processed.The expression of the Orf112 and RstB-His proteins was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cell extracts of VGJφ-infected 569B and E. coli Top 10 transformed with pBAD/Myc-HisC-rstB 9 contained proteins with apparent molecular sizes of 12.7 kDa (Fig. ​(Fig.1)1) and 16 kDa (Fig. ​(Fig.2B),2B), respectively, which are not observed in cells from control cultures. The sizes match those predicted from orf112 (12.72 kDa) (see reference 1) and recombinant rstB-his (16.8 kDa).Open in a separate windowFIG. 1.SDS-PAGE monitoring of the purification process of Orf112 protein. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of non-VGJφ-infected V. cholerae 569B; lane 3, cell extract of VGJφ-infected V. cholerae 569B; lane 4, soluble fraction of the sonicate; lane 5, insoluble fraction of the sonicate; lane 6, precipitate at 30% (NH₃)₂SO₄; lane 7, supernatant at 30% (NH₃)₂SO_4; lane 8, precipitate at 50% (NH₃)₂SO₄; lane 9, supernatant at 50% (NH₃)₂SO₄; lane 10, Orf112 protein electro-eluted after preparative SDS-PAGE.Open in a separate windowFIG. 2.Isolation and purification of RstB-His. (A) Chromatogram on a Ni-CAM HC His tag affinity column. (B) SDS-PAGE monitoring of the purification process of RstB-His. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of uninduced cultures; lane 3, cell extract of expression-induced cultures; lane 4, soluble fraction of the sonicate from expression-induced cultures; lane 5, insoluble fraction of the sonicate from expression-induced cultures; lane 6, soluble fraction of the 8 M urea extract; lane 7, RstB-His eluted from the column.These proteins are not secreted into the growth medium (not shown); thus, they were released from the cells by ultrasonic disruption as previously described (4). V. cholerae was suspended in 15 ml of 20 mM Tris-HCl buffer, pH 7.5, while E. coli cells were suspended in 15 ml of 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. Cell lysates were cleared by centrifugation for 40 min at 9,000 × g and 4°C. SDS-PAGE detected Orf112 protein in the soluble fraction, while RstB-His remained in the insoluble fractions of cell extracts (Fig. ​(Fig.1).1). Subsequently, 569B cell lysate supernatants containing Orf112 protein were fractionated with ammonium sulfate. At 30% ammonium sulfate, several contaminants precipitated but Orf112 protein remained in solution, while at 50% ammonium sulfate, Orf112 precipitated and was recovered by centrifugation. The pellet was washed twice with 50% ammonium sulfate and finally resuspended into 3 ml of 20 mM Tris-HCl buffer, pH 7.5. Removal of excess salt was achieved by gel filtration before the extract was applied to a preparative SDS-PAGE gel. Briefly, a 15% polyacrylamide gel (17 by 19 by 0.5 cm) was run at a constant current intensity of 100 mA and with free voltage at 4°C, until the bromophenol blue dye migrated off the gel. The gels were negatively stained with imidazole-zinc (3), and the Orf112 protein band was identified by comparing bands with the bands of a negative control applied in a neighboring lane of the same gel, where this protein was not visible. The ORF112 protein band was cut from the gel, and the slice was fragmented and introduced into a dialysis bag with a 6- to 8-kDa molecular mass cutoff in 10 ml of 24 mM Tris-HCl-250 mM glycine-0.5% (wt/vol) SDS buffer. The protein was electro-eluted for 5 h at a current intensity of 70 mA and with free voltage at 4°C. Reverse current was applied for 5 min to release membrane-bound proteins, and gel fragments were discarded. The same sample was dialyzed against 1 liter of 0.5 M Tris-HCl, 0.25 M glycine buffer, pH 7.5, for 24 h at 4°C with constant stirring. The dialysis was repeated with 20 mM Tris-HCl, 0.5 M NaCl buffer, pH 7.5, for 24 h. No contaminants were seen when 25 μg of this Orf112 protein-dialyzed extract was checked by SDS-PAGE and Coomassie brilliant blue staining (Fig. ​(Fig.1,1, lane 10).RstB-His protein was recovered from the insoluble fraction of the E. coli lysate by dissolving the lysate in 15 ml of a buffer containing 8 M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 10 mM imidazole, pH 8.0. The mixture was stirred overnight at 4°C and cleared by centrifugation at 9,000 × g for 40 min. The supernatant was applied to a Ni-CAM HC matrix (Sigma), and urea was removed using a linear gradient from 8 to 0 M urea as previously described (5). The presence of 10 mM imidazole in the sample and binding buffer was intended to reduce the level of contaminants bound to the column. Protein was eluted using a gradient of imidazole (10 to 250 mM) in 20 mM sodium phosphate, 0.5 M NaCl buffer, pH 8.0. Fractions were assayed by SDS-PAGE, and those containing the RstB-His protein were pooled according to purity rather than yield. RstB-His protein was obtained with 90% purity (Fig. ​(Fig.2B,2B, lane 7), according to a densitometry scan of Coomassie brilliant blue-stained gels, using a Gene Genius gel documentation system (Syngene Synoptics Ltd., Cambridge, United Kingdom). The gradient-based removal of urea allowed effective solubilization of RstB-His without significant precipitation of protein in the column, as described before (5).Biological activity was assayed by retardation assays of VGJφ genomic ssDNA by 0.5% agarose gel electrophoresis conducted with 20 mM EDTA, 40 mM Tris-acetate buffer. Various amounts of each protein and ssDNA from VGJφ (0.5 μg) mixed in 20% glycerol, 0.25 mM EDTA, 0.3 μM bovine serum albumin, 20 mM Tris-HCl up to a total volume of 40 μl were incubated at room temperature for 30 min and loaded into the gel for analysis. The electrophoresis was run at 100 V and 4°C until colorant exit. Ethidium bromide (1 μg/ml) was used for 30 min to stain DNA bands, which were documented in a Gene Genius gel system (Syngene Synoptics Ltd., Cambridge, United Kingdom).Orf112 protein exhibited DNA retardation activity, showing a high specificity of binding for the circular ssDNA of VGJφ, but was unable to bind double-stranded DNA (dsDNA) (Fig. ​(Fig.3A).3A). However, RstB was able to bind ssDNA as well as dsDNA substrates (Fig. ​(Fig.3B).3B). No retardation was observed with protein preparations from negative controls or when the DNA-protein mixture was inactivated with 1:1 (vol/vol) phenol-chloroform, indicating that the binding activity is intrinsic to the purified proteins.Open in a separate windowFIG. 3.Gel retardation assays of VGJφ-ssDNA by Orf112 protein or RstB-His binding, as measured by 0.5% agarose gel electrophoresis. (A) Binding of Orf112 protein. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lanes 2 to 6; same as for lane 1 plus 1.25, 2.50, 5.00, 10.0, and 20.0 μg of Orf112 protein, respectively; lane 7, same as for lane 6 but treated with phenol-chloroform; lane 8, linearized replicative-form dsDNA of VGJφ; lane 9, same as for lane 8 plus 20.0 μg of Orf112; lane 10, linearized pUC19; lane 11, same as for lane 10 plus 20.0 μg of Orf112. (B) Binding of RstB-His. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lane 2, same as for lane 1 plus 20 μg of RstB treated with phenol-chloroform; lanes 3 to 8, same as for lane 1 plus 0.62, 1.25, 2.50, 5.00, 10.0, and 20.0 μg of RstB-His, respectively; lane 9, linearized replicative-form dsDNA of VGJφ; lane 10, same as for lane 9 plus 20.0 μg of RstB-His; lane 11, linearized pUC19; lane 12, same as for lane 11 plus 20.0 μg of RstB-His.In the case of RstB, which binds to ssDNA and dsDNA substrates, we wanted to rule out the possibility that this effect was caused by the His tail fused to the recombinant protein. For this, we recloned RstB in the same vector as RstB-His but without the His tail. We used the same procedure described above for RstB-His but used oligonucleotides CNC06-171 (see above) and CNC06-172 (5′-TACTGCAGTCAAGATTTAATGGGTTG-3′) for RstB amplification. In this case, CNC06-172 introduced a restriction site for PstI, which was used for cloning into pBAD/Myc-HisC.For purification of this RstB variant, E. coli growth and protein expression induction was done as described for RstB-His. Again, RstB was recovered in the insoluble fraction after cell disruption by sonication. Inclusion bodies were resuspended in 15 ml of 50 mM phosphate buffer, pH 7.7, containing 8 M urea, and after overnight stirring at 4°C, the suspension was cleared by centrifugation (9,000 × g, 40 min). The supernatant was applied to an SP Sepharose fast-flow column (Amersham, United Kingdom), and urea was removed as described above for RstB-His. The RstB that bound to the matrix was eluted using a gradient of 0 to 2 M NaCl and was obtained with about 92% purity (data not shown). RstB without the His tail also showed binding activity toward ssDNA and dsDNA substrates (data not shown), ruling out the possibility that the His hexamer is responsible for the nonspecific binding of RstB-His.Since RstB has affinity for both ssDNA and dsDNA, the possibility exists that this protein simply binds any DNA nonspecifically due to an effect of a charge interaction with the phosphate backbone of DNA. In order to study the effect of the charge in the DNA-binding activity of RstB, NaCl was included in the reaction mixture at a concentration from 0 to 500 mM (Fig. ​(Fig.4).4). As can be seen in Fig. ​Fig.4,4, the retardation activity of ssDNA was only partially inhibited from a starting concentration of 400 mM (Fig. ​(Fig.4A),4A), while the retardation of dsDNA started to be partially inhibited at 300 mM NaCl (Fig. ​(Fig.4B).4B). Since RstB continues to bind at high salt concentrations, which should equilibrate the charge effect, these results indicate that the DNA binding activity is not due to the presence of positively charged amino acids in the protein backbone but rather to the presence of domains that specifically recognize the ssDNA or dsDNA.Open in a separate windowFIG. 4.Effect of salt concentration and nonrelated dsDNA competition on the binding of RstB. (A) RstB binding to phage ssDNA in the presence of 0 to 500 mM NaCl as detected by 0.5% agarose gel electrophoresis. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl, respectively. (B) RstB binding to replicative-form phage dsDNA in the presence of 0 to 500 mM NaCl. Lane 1, 400 ng of dsDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl. (C) DNA-binding competition by RstB. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 and 4, same as for lane 2 plus 500 and 1,000 ng of sheared calf thymus dsDNA, respectively; lane 5, 500 ng of calf thymus DNA (control).We also investigated whether RstB-His has more affinity for the phage ssDNA than for a nonrelated dsDNA; a DNA-binding competition experiment in which the protein was incubated with a constant amount of ssDNA of VGJφ phage and increasing amounts of calf thymus DNA was performed (Fig. ​(Fig.4C).4C). The ssDNA of VGJφ was retarded by RstB-His even in the presence of 500 and 1,000 ng of calf thymus dsDNA (Fig. ​(Fig.4C,4C, lanes 3 and 4). These results indicate that RstB protein has more affinity for the phage ssDNA than for a non-phage-related dsDNA.Until we know more, RstB is the first protein of a filamentous phage which shows affinity for both ss- and dsDNA, at least in vitro. It is possible that RstB needs another protein from the host or the phage itself to recognize the ssDNA in a specific manner, or perhaps the affinity of RstB for both ss- and dsDNA is an intrinsic property of the protein, which is needed on one hand for binding to genomic ssDNA during the rolling circle replication of the phage and on the other hand for holding the hairpin dsDNA secondary structure formed by the phage genome that functions as the site for integration into the bacterial chromosome (12). This hairpin structure is used by XerCD recombinases as a substrate for recombining the phage genome with the bacterial chromosomal dif site (12), and RstB may act jointly with XerCD to achieve integration. This could explain the requirement of RstB for the integration of CTXφ.It is concluded that Orf112 and RstB proteins purified by the protocols described in this paper were biologically active and obtained at a high degree of purity, which paves the way for further characterization of these proteins. The SSB activities of these two proteins are shown for the first time. Consequently, we propose to rename their respective genes gV^VGJφ and gV^CTXφ and their proteins pV^VGJφ and pV^CTXφ, to follow the denomination of genes of canonical phages of the Inovirus genus. A biochemical and chemical-physical characterization of both proteins is in progress and will be published elsewhere. Should the in vitro role demonstrated for these proteins operate in vivo as well, it might explain their role for rolling circle replication and why rstB is required for CTXφ integration.     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.     

Functional unit and product functionality—addressing increase in consumption and demand for functionality in sustainability assessment with LCA

Purpose

The static functional unit definition in the current LCA framework has limitations in addressing the changing product functionality and associated environmental impact of constantly evolving product technologies. As a result, it overlooks the changes in consumer behaviour of increased consumption of products in provided services as well as in growing volumes. This article aims to present a new framework in defining a dynamic functional unit of product technologies that caters for changes in consumer behaviour and growing market.

Methods

A new approach to defining the functional unit is proposed that caters for changes in consumer behaviour and the use of technology from a technical performance perspective. A dynamic approach to definition of the functional unit is proposed that is based on Kano’s model of product function and satisfaction.

Results and discussion

The new approach is demonstrated on a case study in which the analysis of historical data for two TV product technologies—CRT and LCD—is used to show how the total environmental impact is increasing due to the increased functionality which triggers an increase in the volume of the market. Despite the efforts of improving product life cycle design, the society is still faced with increasing environmental impact from the product type overall.

Conclusions

This article presents the challenges of using a static, single functional unit definition in an industrial culture with constant evolution of products that influences usage behaviour and demonstrates the vicious circle of improving product efficiency that leads to further consumption and environmental impact. To address this problem, a new framework of dynamic functional unit definition is put forward for performing comparative LCA to manage the development of product life cycle design that helps keep the total environmental impact of the company’s product portfolio within absolute boundaries.

     

Chromosomal unit fibers in Drosophila

Chromosomal unit fibers consisting of long, regular fibers of about 0.40 m diameter were obtained from disintegrated, isolated chromosomes of two Drosophila melanogaster cell lines. In one cell line with an essentially normal karyotype, three clearly defined size classes of 15, 13, and 11 m length were observed corresponding to the three larger chromosomes of Drosophila. In a cell line carrying an additional translocation between the two largest chromosomes a 19 m fiber derived from the translocation chromosome was observed. Direct determinations of the DNA content per m length of Drosophila unit fibers show that DNA is contracted by a factor of about 1400x in agreement with calculations based on the length of the unit fibers and the known DNA content of the individual Drosophila chromosomes. These findings support our previously proposed model for the unit fiber sub-structure of chromosomes as being derived by a hierarchy of coiling with the corresponding contraction ratios being 7 (100 Å string of nucleosomes), 5 to 6 (250–300 Å thick nucleohistone fiber), and about 40 (unit fiber), resulting in a total contraction of DNA in unit fibers in the order of 1400x.     

基于CVM的海涂湿地生态服务价值的模糊评估模型

正确评估海涂湿地生态服务价值有助于加强人们保护海涂湿地的意识,为海涂湿地围垦生态补偿标准的制定提供依据。视价格"便宜"、"适中"和"昂贵"为模糊集,基于CVM法区间型数据建立了传统模糊统计模型、赋权模糊统计模型和三相划分模型,并以此评价了杭州湾国家湿地公园单位面积年生态系统服务价值。结果显示:3种方法得到的单价分别为10.28、10.38、9.76元m~(-2)a~(-1),三者一致程度较高,与国内沿海湿地价值比较接近。分析了"生态价值"概念客观上的"模糊性"引致模糊数学模型的合理性;采用"橄榄球"式赋权建立赋权模糊统计模型以克服传统模糊统计模型中对区间数据均匀赋权的不合理性,结果在隶属度函数的光滑性和拟合度上好于后者;第三,三相划分模型拟合出3个模糊集的隶属度函数,可以比较相同价格在不同模糊集中隶属度差异。建立的模型对于基于CVM法的生态资源价值评估具有借鉴意义。     

The structure of the adenovirus capsid. II. The packing symmetry of hexon and its implications for viral architecture

The orientation and location of the 240 hexons comprising the outer protein shell of adenovirus have been determined. Electron micrographs of the capsid and its fragments were inspected for the features of hexon known from the X-ray crystallographic model as described in the accompanying paper. A capsid model is proposed with each facet comprising a small p3 net of 12 hexons, arranged as a triangular sextet with three outer hexon pairs. The sextet is centrally placed about the icosahedral threefold axis, with its edges parallel to those of the facet. The outer pairs project over the facet edges on one side of the icosahedral twofold axes at each edge. The model capsid is defined by the underlying icosahedron, of edge 445 A, upon which hexons are arranged. The hexons are thus bounded by icosahedra with insphere radii of 336 A and 452 A. A quartet of hexons forms the asymmetric unit of an icosahedral hexon shell, which can be closed by the addition of pentons at the 12 vertices. Considering the hexon trimer as a complex structure unit, its interactions in the four topologically distinct environments are very similar, with conservation of at least two-thirds of the inter-hexon bonding. The crystal-like construction explains the flat facets and sharp edges characteristic of adenovirus. Larger "adenovirus-like" capsids of any size could be formed using only one additional topologically different environment. The construction of adenovirus illustrates how an impenetrable protein shell can be formed, with highly conserved intermolecular bonding, by using the geometry of an oligomeric structure unit and symmetry additional to that of the icosahedral point group. This contrasts with the manner suggested by Caspar & Klug (1962), in which the polypeptide is the structure unit, and for which the number of possible bonding configurations required of a structure unit tends to infinity as the continuously curved capsid increases in size. The known structures of polyoma and the plant viruses with triangulation number equal to 3 are evaluated in terms of hexamer-pentamer packing, and evidence is presented for the existence of larger subunits than the polypeptide in both cases. It is suggested that spontaneous assembly can occur only when exact icosahedral symmetry relates structure units or sub-assemblies, which would themselves have been formed by self-limiting closed interactions. Without such symmetry, the presence of scaffolding proteins or nucleic acid is necessary to limit aggregation.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

Fluorescence-based mutation detection

Conventional SSCP analysis of DNA amplified by polymerase chain reaction (PCR-SSCP) is one of the simplest and most reliable tools for identifying point mutations, and small insertions or deletions. The sensitivity of the technique is increased by using the Applied Biosystems (ABI) semiautomated DNA sequencer equipped with GENESCAN 672 software for F-SSCP. The four-dye ABI system permits a red dye-labeled internal lane standard to be run in the same lanes as the DNA being examined, leaving three dye colors for labeling DNA of interest. The internal lane standard is used to normalize gels or correct for minor differences in apparent electrophoretic mobility between lanes. Correction for these lane-dependent differences in migration and the capability to stack data from two different lanes on the computer screen makes it possible to detect sequence variants that produce very small mobility shifts. Coelectrophoresis of control and unknown DNA in the same lane, using different dye labels for each, is also helpful for detecting sequence variants that produce small mobility changes. Multiplexing multiple F-SSCP targets in the same lane increases sample throughput.     

Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution

The X-ray structure of a new crystal form of chicken heart muscle citrate synthase, grown from solutions containing citrate and coenzyme A or L-malate and acetyl coenzyme A, has been determined by molecular replacement at 2.8-A resolution. The space group is P4(3) with a = 58.9 A and c = 259.2 A and contains an entire dimer of molecular weight 100,000 in the asymmetric unit. Both "closed" conformation chicken heart and "open" conformation pig heart citrate synthase models (Brookhaven Protein Data Bank designations 3CTS and 1CTS) were used in the molecular replacement solution, with crystallographic refinement being initiated with the latter. The conventional crystallographic R factor of the final refined model is 19.6% for the data between 6- and 2.8-A resolution. The model has an rms deviation from ideal values of 0.034 A for bond lengths and of 3.6 degrees for bond angles. The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution. The crystalline environment of each subunit of the chicken enzyme is different, yet the conformation is the same in each. The open conformation is therefore not an artifact of crystal packing or crystallization conditions and is not species dependent. Both "open" and "closed" crystal forms of the chicken heart enzyme grow from the same drop, showing that both conformations of the enzyme are present at equilibrium in solution containing reaction products or substrate analogues.