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Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins α-lactalbumin, lysozyme, β-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.

The mass transfer properties of polyglycidylmethacrylate–ethylenedimethacrylate monolithic ion-exchangers (convective interaction media disks) were evaluated. As a reference material, the particulate ion-exchanger Source 30 was selected. The model proteins lysozyme, bovine serum albumin, and IgG were loaded at different concentrations and velocities. The mass transfer zones obtained with the monoliths were affected by neither the linear flow velocity nor the protein concentration in the mobile phase. The reduced height equivalent to one theoretical plate (HETP) of monoliths were independent of the reduced velocity. This was not the case for the particulate material.

Purification of green fluorescent protein from a crude solution has been investigated using a combined system of ion-exchange chromatography and size-exclusion chromatography packed into one column of a continuous annular chromatograph. Performing the chromatography with two different sorbents packed into a single column reduces transfer losses and the hold steps between the two separation steps can be eliminated. Appropriate running conditions for the continuous separation mode were achieved by searching for optimal ones in conventional batch-wise operation. Green fluorescent protein was expressed in Saccharomyces cerevisiae, enzymatically lysed and the extract was used as the feed stock. Superdex 200 prep grade was packed into an annular chromatograph (outer diameter: 15 cm, inner diameter: 14 cm) and on top a 1.5 cm layer of the anion-exchange sorbent Source 30 Q. Green fluorescent protein was enriched by the anion-exchange resin and further purified by the size-exclusion gel. Purity was analyzed by so...

The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.

A peptide screened from a combinatorial peptide library with the sequence EYKSWEYC performed best as a ligand for affinity chromatography of human blood coagulation factor VIII (FVIII). With this peptide immobilized on monolithic CIM columns via epoxy groups we were able to capture FVIII from diluted plasma. Rational substitution of amino acids by spot synthesis revealed that lysine and cysteine can be exchanged for almost all other proteinogenic amino acids without loss of affinity to FVIII. This offers the possibility of site-specific attachment via either one of these residues or the N- or C-terminus. The aliphatic positions O5 (tryptophan) and O7 (tyrosine), together with the charged position O6 (glutamic acid), seem to form the core of the binding unit. In the positions with aliphatic amino acids, substitution by tyrosine or phenylalanine, and in the positions with charged amino acids, substitution by aspartic acid or lysine, preserved the affinity to FVIII. The functionality of the selected peptides was confirmed by affinity chromatography. Selective binding and elution could be achieved.

Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so-called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half-life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core-fucosylation of the N-glycan and aggregation the effects are clear and should be monitored, but with others such as C-terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl 2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup until attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.

International audience; Cation exchange chromatography has been routinely used for the quantification of monoclonal antibody (mAb) charge heterogeneity. A previously developed method utilizing pH gradients for the elution instead of salt gradients was validated according to current guidelines proposed by the ICH. The linearity, stability, accuracy, precision and the lower limit of quantification have been determined, using pure charge variant standards. The method is valid for the quantification of mAb samples with a charge heterogeneity between 1% and 50%. Three different approaches to obtaining pure standard material for the validation of bio-analytical methods for the quantification of charge heterogeneity of IgG are presented. These methods are based on salt gradient elution, pH gradient elution and displacement in cation exchange chromatography.

Seven porous chromatographic columns, termed monoliths, and seven nonporous sheets were produced from polymethacrylates. Their surfaces were activated by different densities of butyl and phenyl ligands. We determined the retention times of highly dilute molecular probes in monoliths and accessed contact angles of pure molecular probes of sheets. We calculated surface energies for both systems. We applied theories of Young, Dupre, and van Oss and compared the results of both types of experiments with respect to Lifshitz–van der Waals and Lewis acid and Lewis base contributions and find agreement but an additive constant.

Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation.

Highlights • Size exclusion chromatography is a general method for nanoparticles separation. • The method is suited for purifying large quantities of protein covered nanoparticles. • Nanoparticles and proteins can be continuously separated by simulated moving bed chromatography. • Particle and protein fractions reached a purity of > 90%. • Reuse of buffer and unbound protein was possible using tangential flow filtration., For scale up and efficient production of protein loaded nanoparticles continuous separation by size exclusion chromatography in simulated moving bed (SMB) mode helps do reduce unbound protein concentration and increase yields for perfectly covered particles. Silica nanoparticles were loaded with an excess of beta casein or bovine serum albumin (BSA) and the loaded particles purified by size exclusion chromatography using Sephacryl300 as stationary phase in a four zone SMB. We determined our working points for the SMB from batch separations and the triangle theory described by Mazzotti et al. with an SMB setup of one Sephacryl300 26/70 mm column per zone with switch times of 5 min for BSA and 7 min for beta casein. In the case of BSA the Raffinate contained loaded nanoparticles of 63% purity with 98% recovery and the extract was essentially particle free (95% purity). We showed that the low purity of the Raffinate was only due to BSA multimers present in the used protein solution. In the case of beta casein where no multimers are present we achieved 89% purity and 90% recovery of loaded nanoparticles in the Raffinate and an extract free of particles (92% purity). Using a tangential flow filtration unit with 5 kDa cutoff membrane we proved that the extract can be concentrated for recycling of protein and buffer. The calculated space–time-yield for loaded nanoparticles was 0.25 g of loaded nanoparticles per hour and liter of used resin. This proves that the presented process is suitable for large scale production for industrial purposes.

A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.

Background Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats. Methods In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen. Results This study led to the identification of 2′-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2′-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships. Conclusions These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.

The remodeling of lipids in adipocytes is an interesting property of compounds with the ability to ameliorate metabolic syndrome. In this study, the binding affinity of pomegranate to the peroxisome proliferator-activated receptor γ (PPARγ) was compared with plant extracts that are commercially on offer as anti-diabetic dietary supplements such as cinnamon, purslane, grape wine, bitter melon, Kothala himbutu, and Coccinia indica. This receptor is a drug target for diabetes type 2. One of the most potent pomegranate extracts was selected and tested in a murine 3T3-L1 cell system to examine effects on adipocyte differentiation. Pomegranate extracts had an extremely high binding affinity for PPARγ, which was several fold higher in comparison to other tested extracts. Pomegranate extract also modulated adipocyte differentiation, which resulted in lipid remodeling and the formation of micro-lipid droplets, which increases total lipid droplet surface area to volume ratio and enables an easier lipid breakdown due to lipolysis. The results corroborate the hypothesis that pomegranate has a benevolent impact on obesity and diabetes type 2. As modulator of PPARγ and the adipose tissue, pomegranate is a promising plant for the amelioration of metabolic syndrome.

Raloxifene represents a potent compound for the prevention and treatment of osteoporosis and cardiovascular disease in postmenopausal women. Raloxifene exhibits targeted antiestrogenicity in breast and uterus, but acts as an agonist in bone and liver. This synthetic selective estrogen receptor modulator binds both estrogen receptors α and β. The molecular mechanisms by which raloxifene exerts agonistic or antagonistic activity are still not resolved. Therefore, the binding behavior of raloxifene to estrogen receptors and its effects on DNA binding and transactivation were studied. The equilibrium binding affinity of raloxifene by displacing radiolabeled 17β-estradiol exhibited a similar affinity behavior to that of its natural ligand. Using BIACORE technology with an immobilized estrogen response element, we showed that 17β-estradiol and raloxifene increased the binding of estrogen receptor α to the DNA, suggesting a ligand-dependent dimerization. The influence of the ligands to the binding of estrogen receptor β was lower. We may conclude that unliganded estrogen receptor α binds as a monomer whereas in the presence of 10 −8 M 17β-estradiol or higher, homodimers are formed that interact with the estrogen response element. Transactivation studies in a yeast reporter system in a ligand-dependent manner resulted in a similar potency of raloxifene to estrogen receptor β compared to the control testosterone. Subeffective doses of raloxifene combined with 17β-estradiol did not shift the efficiency, whereas saturating concentrations of 17β-estradiol combined with increasing concentrations of raloxifene altered the response induced by 17β-estradiol. In this pure system, the antagonistic activity of raloxifene could not be detected as was expected by the results from ligand competition analysis.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.

The FLAG (Sigma, St. Louis, MO, USA) peptide is a frequently used hydrophilic and immunogenic fusion tag which was specifically designed to facilitate rapid purification by immunoaffinity chromatography. The monoclonal antibody M1 recognizes the free N-terminus of the peptide tag in a calcium dependent manner. Dissociation of the complex can be performed by the addition of chelating agents such as EDTA. This effect can be exploited for immunoaffinity purification of FLAG-tagged fusion proteins. Kinetic information obtained from monitoring interactions in real-time measurement (Biacore 2000) using surface plasmon resonance as detection principle did not show any difference for association and dissociation rate constants in the presence (k(a) = 3.03 x 10(3) M(-1) k(d) = 1.25 x 10(-3) s(-1)) and in the absence of Ca2+ (k(a) = 3.59 x 10(3) M(-1) s(-1), k(d) = 1.16 x 10(-3) s(-1)). These findings corroborate the reports from Mol. Immunol. 33 (1996) 601-608 describing similar binding analyzed by enzyme-linked immunosorbent assay experiments. These investigations are in contrast to the observations in immunoaffinity chromatography with immobilized anti-FLAG antibody M1.

Transport rates (permeability) and ideal separation factors for several gas pairs through dense polyaniline membranes are reported. The ideal separation factors for all gas pairs tested were found to be independent of the polyaniline membrane thickness whereas the permeability of the single gases showed significant variations. Both dedoped and redoped films (film thickness between 9 and 67 μm) were studied. The highest selectivities α (A/B) found were 7.6 for the gas pair H 2 /CO 2 in the case of the dedoped membrane and 10 for the gas pair H 2 /CO 2 , 6 for O 2 /N 2 and 200 for H 2 /N 2 in the case of the redoped membrane. Statistical analysis of a large number of membranes allowed the critical comparison with results obtained by other groups. Comparison with other membrane materials shows that an approximately sixfold enhancement of the respective separation factors is possible for gas pairs containing hydrogen. Similar separation factors are observed for the gas pairs CO 2 /O 2 , CO 2 /N 2 and N 2 /O 2 . Membranes for which Knudsen diffusion was observed exhibited regularly distributed micropores (400 nm diameter).

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 microl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.

Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The affinity-purified and deglycosylated protein is a tool for further biochemical and biophysical characterization of SLAM.

It has been suspected that beer drinking may change the hormonal status of men caused by phytoestrogens. Five different Austrian lager beers have been investigated for estrogenic activity by a yeast two-plasmid system harboring the human estrogen receptor α, after concentration by solid phase extraction. The beer concentrate was further fractionated by reversed phase HPLC, and then the fractions were characterized by the biological assay and GC-MS. The most potent fraction did not contain a known phytoestrogen. The total activity corresponded to an average of 43 ng of 17β-estradiol/L of beer. It was concluded that the human health hazard of beer drinking originating from compounds activity on the estrogen receptor α is negligible. Keywords: Phytoestrogens; beer; yeast; isoflavonoids

Monoliths are stationary phases cast as a continuous medium which are interlaced by flow channels ramified with micropores. Pulse response experiments with bovine serum albumin as a model protein were applied for testing polymethacrylate-based monoliths, resulting in peak broadening that practically was not influenced by the chromatographic velocity. An empirical model was developed to describe peak broadening, allowing a term to account for the pore convection and a term for the pore diffusion. A diffusional distance lower than 10 nm was estimated. This corresponds to values observed with monodisperse 1-microm particles. Systematic investigations by changing the response time of the detector showed that the full potential of the monoliths could not be exploited, since the currently available chromatography systems are the limiting factor regarding the speed of data acquisition and virtual peak broadening by the infinite length of the detector. Inertia of the liquid and synchronization between liquid handling and electronic control introduced an additional disturbance. At the lowest possible response time, reliable peak data could be obtained up to a velocity of 35 cm/min. The pressure drop along the continuous bed was much smaller compared to a conventionally packed bed. Different flow patterns and significantly reduced eddy vortexes may be responsible for the high specific permeability.

Theoretically, in fixed-bed chromatography an infinite separation time or volume is available. In annular chromatography the separation time is limited by the time required for a single rotation. In a chromatograph with a smaller annulus than the theoretical one, the equilibration zone will overtake the feed zone. The required separation time is determined by a number of steps such as equilibration, loading, washing, elution, regeneration and the retention of the solute. A mathematical model has been developed to estimate the minimum radius of an annular chromatograph. The minimal geometry of an annular chromatograph was calculated for a model system immunoglobulin and bovine serum albumin.

Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.

The background of agonist/antagonist behaviour of the non-steroidal antiestrogen tamoxifen is still not fully understood. Depending on cell type, its activities range from full agonistic to antagonistic in different tissues. We investigated the transactivational properties of tamoxifen in a basic yeast model system which reconstitutes ligand-dependent human estrogen receptor-alpha (hER alpha) gene activation. Tamoxifen exerted low agonist activity in this system compared to 17 beta-estradiol (E2). Efficiencies and potencies of several isomers were calculated by fitting experimental data with a logistic dose-response function. Cis-, trans- and cis-transtamoxifen and trans-4-hydroxytamoxifen (4-OHT) showed comparable efficiencies and potencies in yeast. When subeffective doses of trans-, cis-/trans-, or trans-4-OH tamoxifen were combined with increasing 17 beta-estradiol concentrations, even a synergistic increase in efficiencies could be observed. Interestingly, the cis-isomer did not show this synergistic effect. Mutation of the N-terminus of the estrogen receptor changed the transactivational behaviour of tamoxifen and abolished the synergistic action with 17 beta-estradiol. Except for 4-OHT, the potencies of the investigated isomers, defined as ligand concentrations with half-maximal response, highly correlated with the binding affinities to hER alpha. Therefore, cis-, trans-, and cis-/trans-tamoxifen could be regarded as full agonists in yeast, while 4-OHT was regarded as a partial antagonist in yeast. Furthermore, these results indicate that the functional difference between trans-tamoxifen and trans-4-OHT is not due to their different affinities for the receptor protein.

Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.

A rapid, simple in vitro test system for high-throughput screening of peroxisome proliferator-activated receptor (PPAR) gamma agonists would be of interest for testing new antidiabetic drugs, alternative medicine, or environmental samples. A yeast two-hybrid assay based on the ligand-dependent recruitment of the coactivator CBP (CREB-binding protein) was constructed. In this system PPARgamma was constitutively activated and the signal was not further increased significantly by adding agonists. In yeast we identified oleic acid as a putative endogenous ligand. Furthermore yeasts seem to lack regulatory mechanisms present in mammalian cells. Mammalian systems are an alternative for screening PPARgamma agonists.

Estrogen receptor-α is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-α has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293] in a 14 l stirred tank reactor. The yeast extract contained 2–4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17β-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5–10-fold in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17β-estradiol 17-hemisuccinate–bovine serum albumin–Sepharose. The receptor could be kept in its native state, although saturated with 17β-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.

A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both IM + H] + ions by MALDI and both [M + H] + and IM + 2H] 2 + ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN] + ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.

Preparations of clotting factor VIII (FVIII) are complex protein mixtures, regardless of whether they are made by genetically engineered cells or isolated from human plasma. Commercially available FVIII preparations contain either von Willebrand factor (vWF) or human serum albumin (HSA), both of which function as stabilizers. Apart from these, the preparations contain other plasma proteins as impurities. Such protein mixtures were analyzed with SDS–PAGE and 2D-electrophoresis. Single bands and spots were identified by immunoblot using monoclonal and polyclonal antibodies. The aim was to detect every preparation which has an increased level of FVIII cleavage products. Chromatographic methods and SDS–PAGE under reducing and non-reducing conditions usually do not show any differences between single preparations. Only by a combination of separations under non-reducing and increasingly reducing conditions cleavage products of FVIII can in some cases be detected in immunoblot. In 2D electrophoresis the detection of proteins with high molecular masses was achieved by applying a mixture of thiourea and urea. The spots in 2D-electrophoresis of plasma-derived FVIII preparations have been identified as FVIII and vWF, but also as impurities, chiefly from fibrinogen. It was shown that especially 2D-electrophoresis and immunoblot can be used for better characterization of FVIII concentrates. The 2D protein maps of FVIII concentrates will be an efficient tool for quality control of such products.

Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media. A fractionation scheme for the economically interesting proteins, such as IgG, lactoferrin and lactoperoxidase, based on cation exchangers was the goal of our investigations. A chromatographic process was developed where alpha-lactalbumin passes through the column and separation of the desired proteins is achieved. Four different cation-exchange media (S-HyperD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (S) and Macro-Prep High S Support) were compared in regard to their dynamic binding capacity for IgG and their different elution behaviours when sequential step gradients with NaCl buffers were applied. Peak fractions were analyzed by size-exclusion chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Lactoperoxidase activity was monitored by the oxidation of o-phenylenediamine. In order to explain the different resolution behaviours, isocratic runs with pure standards of whey proteins were performed. The k' values were calculated and plotted against salt concentration. Fractogel EMD had the highest binding capacity for IgG, 3.7 mg/ml gel at a linear flow-rate of 100 cm/h, but the resolution was low compared to that with the other three media. S-Hyper D and S-Sepharose FF showed lower capacities, 3.3 and 3.2 mg/ml gel, respectively, but exhibited better protein resolution. These effects could be partially explained by the k' versus salt concentration plots. The binding capacity of Macro-Prep S was considerably lower compared to that of the other resins investigated because its selectivity for whey proteins was completely different. S-Sepharose FF and S-Hyper D combine relatively high dynamic capacity for IgG and good resolution. Compared to studies with standard proteins, such as 100 mg/ml bovine serum albumin for S-Hyper D, their binding capacities were very low. Even after removal of low-molecular-mass compounds, the capacity could not be improved significantly. The running conditions (low pH) were responsible for the low protein binding capacity, since low-molecular-mass compounds in the feed do not compete with the adsorption of whey protein. The dynamic capacity did not decrease to a large extent within the range of flow-rates (100-600 cm/h) investigated. The dynamic capacity of HyperD and Fractogel was at least five times higher when pure bovine IgG was used for determination. In conclusion, S-Sepharose FF, S-Hyper D-F and Fractogel EMD SO3- 650 (S) are considered as successful candidates for the large-scale purification of bovine whey proteins.

In preparative chromatography of proteins and other biopolymers, elution is frequently achieved by a stepwise or continuous change of the mobile phase composition, generally denoted as step or linear gradient. The gradient is formed by inline mixing of two aqueous buffers. For prediction of peak profiles, examination of the reliability of blending devices and remote control of chromatography controllers a model would be advantageous. This would be most beneficial for ion-exchange chromatography and hydrophobic interaction chromatography where aqueous buffers must be blended. Here different salt concentrations are commonly blended. The model is able to describe the delay of onset and the transition period for step gradient. We developed a model based on the simple continuously stirred tank reactor model (CSTR) which has been modified to include logistic growth. Two parameters have to be estimated; parameter a is a lumped parameter related to the exponential time constant and Gaussian broadening. The second parameter, denoted b, describes the delay inherent to the system. This novel model is compared to a model based on the convolution of a step input with a Gaussian broadening and an exponential decay. The predicted values of both models are similar, when compared to experimental data obtained from inline mixing of two salt buffers. The logistic growth modified CSTR model exhibited higher residuals than the convolution model. The beginning of the gradient cannot be approximated in a smooth fashion, since the applied equation contains a sharp inflection. The current model's application is limited to the blending of salt buffers; pH is not a consideration of this model.

Practical considerations lead to the use of small columns in preparative chromatography. In these applications extra column effects may overwhelm all broadening effects. The influence of the extra column effects on the band broadening and migration is demonstrated by simulation. The model assumes linearity of extra column effects. Consequently, these effects could be described by the convolution of the initial applied band width, an exponential decay and Gaussian broadening. An attempt has been made to discriminate between the column, pre- and postcolumn broadening.

Recombinant antibodies with high isoelectric point are frequent since most of them are constructed from the same framework. Classically, cation exchange chromatography is used as a standard method for the determination of antibody charge heterogeneity. In contrast, in this study highly linear pH gradients were achieved by keeping the buffering capacity over the length of the gradient constant. The buffering compounds were selected to be unretained on the column and their respective concentration was adjusted in the start and end buffer of the pH gradient to achieve constant buffering capacity. This helps conserve linearity and stability of the gradient. The method allows quantification of charge variant distribution and the determination of chromatographic isoelectric point. To demonstrate the effectiveness of this novel method, a ProPac WCX-10 column was used to separate isoforms of trastuzumab biosimilar antibodies. Effects of pH gradient linearity and of varying the analytical amount of sample on the separation are shown.

The latest ESBES special issue on "Biochemical Engineering Sciences" is edited by Prof. Guilherme Ferreira (Chairman, ESBES) and Prof. Alois Jungbauer (co-Editor-in-Chief, Biotechnology Journal). This special issue comprises the latest research in biochemical engineering science presented at the 9(th) ESBES Conference held in Istanbul, Turkey in 2012.

Ethnopharmacological relevance Eriosema laurentii De Wild (Leguminosae) is a medicinal plant used in West and Central Africa for different diseases. In Cameroon, this plant is used as a treatment for infertility, and various gynecological and menopausal complaints. However, despite this use as a natural remedy, the biological activity of Eriosema laurentii has not been studied until now. Aim of study In order to determine the potential use of this plant in gynecological conditions/disorders, we evaluated the estrogenic properties of a methanol extract of its aerial parts and its ability to prevent different menopausal health problems induced by bilateral oophorectomy. Material and methods Two approaches were used. In vitro , recombinant yeast systems were applied, featuring either the respective human receptors (ERα, AR, and PR) or into chromosome III integrated human aryl hydrocarbon receptor (AhR) and the respective reporter plasmid. In vivo , the investigation was carried out using the 3 days uterotrophic assay and 9 weeks oral treatment in ovariectomized rats. Results The results showed that the methanol extract of the aerial parts of Eriosema laurentii transactivated the estrogen receptor-α and displayed AhR agonistic activity but was neither androgenic nor progesteronic. In rats, the extract did not induce endometrium proliferation either in the 3-day or the 9-week treatment regimens, but induced vaginal stratification and cornification, prevented loss of femur bone mass, increased high density lipoprotein cholesterol (HDL-C), and reduced total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), TC/HDL-C ratio, LDL-C/HDL-C ratio and the atherogenic index of plasma (AIP). Conclusion These results suggest that the methanol extract of the aerial parts of Eriosema laurentii does not seem to have an undesirable influence on the endometrium but might prevent vaginal dryness and bone mass loss and improve the lipid profile.

A reversed phase high pressure liquid chromatography method was developed for determination of in vitro refolding and cleavage kinetics for the N pro autoprotease fusion peptide EDDIE-pep6His using a TSK Super-Octyl column with a segmented acetonitrile gradient. Self-cleaving fusion proteins such as N pro autoprotease fusion proteins consist of the single autoprotease N pro and a target peptide or a target protein as fusion partner. Hence, three protein species are present after self-cleavage: the target peptide or protein, the single N pro autoprotease and, in case of incomplete cleavage, residual N pro fusion protein. Thus, for an accurate analysis the method must be standardized for three components in the presence of host cell impurities. For method validation, protein standards of EDDIE-pep6His and the single N pro autoprotease EDDIE were prepared from inclusion bodies (IBs) by ion exchange, immobilized metal ion affinity, size exclusion, and reversed phase chromatography. A linear correlation was obtained for EDDIE-pep6His and EDDIE in the range from 95 to 730 μg/ml with a lower limit of quantification (LLOQ) and a lower limit of detection (LLOD) of 34.5 and 11.4 μg/ml, respectively, for EDDIE-pep6His and 39.6 and 13.1 μg/ml, respectively, for EDDIE. Finally, a fully automated batch refolding of EDDIE-pep6His from IBs was performed to demonstrate the applicability of this method. It was shown that the initial EDDIE-pep6His concentration in the refolding solution decreased from 194.3 to 83.8 μg/ml over a refolding time of 385 min resulting in a final refolding and cleavage yield of 50%.

Inclusion bodies (IBs) were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the IB solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the IBs homogeneously suspended, and additional power input will not improve the process. The high-throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale.

Continuous manufacturing has been applied in many different industries but has been pursued reluctantly in biotechnology where the batchwise process is still the standard. A shift to continuous operation can improve productivity of a process and substantially reduce the footprint. Continuous operation also allows robust purification of labile biomolecules. A full set of unit operations is available to design continuous downstream processing of biopharmaceuticals. Chromatography, the central unit operation, is most advanced in respect to continuous operation. Here, the problem of ‘batch' definition has been solved. This has also paved the way for implementation of continuous downstream processing from a regulatory viewpoint. Economic pressure, flexibility, and parametric release considerations will be the driving force to implement continuous manufacturing strategies in future.

A chromatographic process based on monoliths for purification of infective baculovirus without prior concentration step has been established. Baculovirus produced in Spodoptera frugiperda cells (Sf-9) were harvested by centrifugation, filtered through 0.8 μm filters and directly loaded onto radial 1 mL anion exchange monoliths with a channel size of 1.5–2.0 μm operated at a volumetric flow rate of one bed volume per minute. Optional an epoxy monolith was used as pre-column to reduce interfering compounds and substances influencing the capacity of anion exchange monoliths for baculovirus infectious virus could be eluted with a step gradient at salt concentrations of 440 mM NaCl. Recovery of infectious virus was highly influenced by composition and age of supernatant and ranged from 20 to >99% active baculovirus. Total protein content could be reduced to 1–8% and DNA content to 38–48% in main virus fraction. Infective virus could be 52-fold concentrated within 20.5 h and simultaneously an 82-fold volume reduction was possible when loading 1150 mL (2.1 × 10 8 pfu/mL) onto 1 mL scale support.

A new assay to measure cell proliferation by turbidimetric evaluation of cultures in specially designed microtiter plates was set up. The IL-2-dependent proliferation of CTLL-2 cell line was used as a model. The novel microtiter plate detection system, the General Cell Screening System (GCSS) was used for the evaluation of the cell proliferation assay. The test system utilizes the population growth rate (mu) of the cells. The tests were performed in specially designed microtiter plates which were read in a scanning spectrophotometer. These results were compared with colorimetric assays, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as substrates. In contrast to conventional methods, the presently described system is not an end-point method, this means that cell growth can be observed over a certain period and is not limited to a single moment during the period of cell growth. Another advantage is the reduction of manual manipulations and the avoidance of toxic or radioactive substances and organic solvents. Therefore, a higher number of samples can be analyzed compared to other methods. The range for detection was 0.015-10 units IL-2 per well, and the accuracy is in the range of < 0.005 standard deviation.

The distribution coefficient (K) of a protein in ion-exchange chromatography is dependent on the protein and salt concentration in the mobile phase. Conventional isotherms only describe the relationship between the mobile phase and stationary phase concentrations of the protein, the influence of salt being neglected. In this paper, the dependence of K on salt is described by transition functions [ϕ(I)] such as logistic dose response and hyperbolic tangent functions. The relationship between K and protein concentration is described by conventional isotherms; the K value is considered as the first derivative of the isotherm, or as the ratio between concentrations in the mobile and stationary phases. The stationary phase concentration can be expressed as a function of the mobile phase concentration. Combinations of these functions with transition functions can be used to approximate the binding characteristics of a protein dependent on protein and salt concentration.

Recent advances in mathematical modeling of protein chromatography have focused on two areas: intra-particle transport and interactions with the sorbent surface. The influence of intra-particle transport has been investigated critically and new theories for intra-particle convection and diffusion have been developed. Careful modeling of so-called ‘perfusion’ chromatography has also provided insight into conventional protein chromatography. Novel experimental methods have been designed to verify or reject the above theories. The adsorption of proteins has been more rigorously investigated, and thermodynamic aspects, protein—protein interaction and the availability of binding sites have been incorporated into existing theories. Finally, algorithms for the optimization of operational variables, such as gradient shape, flow rate, and column utilization, have been incorporated into strategies for rational process design. These strategies use existing or modified mathematical models for the description of migration of a protein zone through a chromatography column.

The expression height equivalent to one theoretical plate (H) is a quality parameter in (process-) chromatography for column packing and particle size distribution of resins. It is found that the value of H, ubiquitously known and accepted by chemical engineers and scientists, is heavily dependent on determination conditions. Determinations of H from pulse experiments and frontal analyses are inconsistent for the three chromatographic resins tested. Varying mathematical methods influence H as well as the salt concentration of the eluent buffers. The biggest difference is found when comparing the H-values of one resin ; the difference, determined by using different methods, was in the range of 2 orders of magnitude. Due to these results, the user of chromatographic resins is advised to interpret H-values supplied by manufacturers carefully and to take into account the dependency of the results on the methodology and conditions of determination.

Complications of DNA clearance in protein chromatography using the conventional methodology of spiking experiments are reported. Protein A affinity chromatography demonstrated this complications in a small scale experiment. A concentrated hybridoma culture supernatant was spiked with DNA extracted from hybridoma cells fed with [3H]thymidine. Protein A affinity chromatography was subsequently carried out. The column effluent was collected in fractions, and each fraction was analyzed for radioactivity and IgG levels. A substantial amount of DNA was eluted before the main IgG peak. Frequently a small peak is observed in front of the main peak in protein chromatography. This phenomenon can be explained by either displacement effects, or incomplete washing, or hysteresis during the adsorption and desorption conditions. Fractionation at the beginning of elution is critical to the maintenance of a high standard protein purity.

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-1 fermentor with 14-1 net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-β- d -thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.