Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis.

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4-6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.

Characterization and application of strong ion-exchange membrane adsorbers as stationary phases in high-performance liquid chromatography of proteins.

Filtration membranes carrying strong cation- or anion-exchange groups on their surface were evaluated for their potential as membrane adsorber stationary phases in the high-performance liquid chromatography of proteins. The membranes are commercially available and can be obtained inserted into ready-to-use filter holders. Owing to their thinness (170-190 microns), the pressure drop of the membranes is extremely low. Flow-rates of up to 65 ml min-1 per unit became thus possible. The low pressure drop of a single membrane layer also permitted an effortless scaling up, as a stack of several membranes or filter units could be used, if necessary. Sample distribution, protein binding capacity, elution conditions, separation efficiency and recovery were investigated as a function of the flow-rate. The time required for the separation of certain protein mixtures could be reduced to less than 1 min. Appropriate conditions were defined for the separation of human serum and for the isolation of subtilisin Carlsberg and beta-galactosidase from cell culture supernatants.

Studies of complexes between proteases, substrates and the protease inhibitor alpha 2-macroglobulin using capillary electrophoresis with laser-induced fluorescence detection.

Capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection is shown to constitute a unique technique for the investigation of the interaction between proteases, protease inhibitors and substrates. Under optimized analysis conditions, the formation of a complex between FITC-labelled proteases such as trypsin, plasmin, alpha-chymotrypsin and the (unlabelled) protease inhibitor alpha 2-macroglobulin was studied. This is not possible with UV detection, since under such conditions the complex cannot be distinguished from the unreacted protease inhibitor. Low ratios of FITC bonded to the proteases further complex formation, while high ratios often prevent the reaction. Complex formation shows a strong dependence on the incubation conditions (pH, salt concentration, temperature, incubation time). Once formed, however, the complexes are stable under CZE conditions (e.g., a pH of the electrophoresis buffer of 10.5) for at least 30 min. Treatment with sodium dodecyl sulfate (5 min at 90 degrees C or 30 min at 75 degrees C) does not destroy the complexes, whereas treatment with mercaptoethanol (reduction of disulfide bonds) eliminates the peak from the electropherogram. Both findings argue for the formation of a covalent bond between the protease and the inhibitor during complex formation. Since the reaction of the proteases with alpha 2-macroglobulin does not involve the binding site of the former, a residual proteolytic activity is still observed in the ensuing complex. The extent of the inhibition of the remaining trypsin activity in a trypsin--alpha 2-macroglobulin complex was established to depend on the molecular mass of the second trypsin inhibitor.

Rapid capillary gel electrophoresis of proteins.

The rapid separation of sodium dodecyl sulphate-protein complexes according to their molecular masses (M(r)) by capillary gel electrophoresis is described. Using commercial equipment, standard proteins with M(r) in the range 29,000-97,400 were resolved to the baseline in less than 2 min by utilizing a separation distance of 7 cm. A linear relationship between migration time and log M(r) was found and rapid determination of the molecular mass of light and heavy chains of human immunoglobulin G is reported. The results are compared with applications using longer separation distances, showing that rapid and efficient analysis and adequate resolution can be obtained by using short separation distances.

High-performance capillary electrophoresis of human serum and plasma proteins.

In recent years, capillary electrophoresis has proved extremely useful for separation of biomolecules such as nucleotides, amino acids, peptides, and proteins. The advanced instrumental design of commercial CE equipment has offered several advantages to conventional electrophoresis. Precise sample application, temperature regulation, and stable, reproducible instrument control with respect to voltage, current, pressure, and run-to-run reproducibility is routinely achieved in contemporary commercially available equipment. Based on these instrumental features, CE is already used as a routine method for the analysis of small molecules. In contrast, serum and plasma proteins represent a difficult challenge for capillary electrophoresis. Routine analysis of serum and plasma proteins is reduced to the application of capillary zone electrophoresis, which proved to be a reliable method for rapid screening of whole serum, purity control of serum proteins and fractions, as well as on-line quantification of the components. With regard to the simplicity of the method and the low cost of operation, CZE proved to be an attractive alternative to comparable conventional methods. Other separation techniques, such as CITP, CIEF, and CGE, provide higher resolution of the sample proteins than that provided by CZE. Furthermore, information about physicochemical properties of the analyte, such as isoelectric point or molecular weight, can be gained by CIEF or CGE. Even though this information is essential for protein analysis and characterization, these methods are not commonly used at present because reliable methods for controlling the EOF are not commercially available. Coated capillaries are expensive, and it is still difficult to purchase a set of coated capillaries with identical properties. As long as the reproducibility from capillary to capillary cannot be guaranteed, the high resolving methods will not be applied in routine analysis. The development of reliable regulation of the EOF, either by an appropriate column material or coating or an external field control, will certainly lead to a breakthrough for the capillary electrophoresis of human serum and plasma proteins.

Analysis and evaluation of filter cartridge extractables for validation in pharmaceutical downstream processing.

This paper describes a comprehensive approach for the extractables analysis of filter cartridges used in pharmaceutical production processes. For the extraction of the cartridges, two model solvents (water and ethanol) and worst case conditions are used. The extracts of 8 cartridges from various filter manufacturers are analysed both in the original and the concentrated form to gain data about the high and low concentrated contaminants, implementing standard analytical techniques such as GC-MS, RP-HPLC, GPC and FT-IR. Degradation products and additives of the filter cartridge polymers are found to be the source of most of the extracted compounds. The concentrations of extractables are determined in the range of ppm to ppb per cartridge under the applied extraction conditions. The effect of rinsing the cartridges prior to use was studied, showing a reduction of the extractables even after small flush volumes.

Comparison of membrane adsorber (MA) based purification schemes for the down-stream processing of recombinant h-AT III.

Several multistage chromatographic separation schemes based on Membrane Adsorbers as stationary phases were designed for the isolation of recombinant human Antithrombin III from supernatants of baby hamster kidney cell cultures. The Antithrombin III concentration of the culture supernatants varied between 6 micrograms/ml and 14 micrograms/ml. The culture media contained 10% foetal calf serum. The concomitant overall protein concentration ranged from 5.4 mg/ml to 6.2 mg/ml, with bovine serum albumin constituting approximately 60%. Strong cation and anion exchanger, Heparin-, and Cibacron Blue Membrane Adsorber were used. While Heparin-Membrane Adsorber were found to isolate and concentrate the Antithrombin III efficiently, the removal of the major bovine serum proteins (albumin, transferrin, immunoglobulins) required a multistage process. By using a sequence of ultrafiltration, diafiltration, Cibacron Blue, anion exchanger, and Heparin-Membrane Adsorber, an electrophoretically pure Antithrombin III could be obtained. Subsequent high sensitivity gel immunoelectrophoresis proved the isolated protein free of bovine IgG and bovine transferrin, while approximately 2% serum albumin could still be discerned. A reduction of the serum content (3%) allowed the isolation of high purity Antithrombin III (> 99.9%), however, the product's specific activity was halved. The Down-Stream-process was designed and optimised for a mobile phase flow rate of 2 ml/min (0.12 l/h). With the exception of the final Heparin affinity step, however, flow rates of up to 4.8 l/h could be used without adverse effect on the final purity, with a concomitant increase of the throughput. Batches of up to 10 1 cell culture supernatant were processed in an automated procedure.

Evaluation of affinity filters for protein isolation.

Affinity filters were investigated for their potential in the recovery of proteins from complex samples. The experiments covered membranes carrying high and low molecular weight affinity ligands as well as group and substance specific ones. For the ready-to-use affinity filters the specific protein binding capacity was determined and compared to that of the respective Sepharose affinity gels (Pharmacia). In the case of the pre-activated membranes the influence of the coupling chemistry on the affinity mediator concentration and the protein binding capacity were considered in the study. In the case of low molecular weight ligands (e.g. Cibacron Blue, Heparin) either type of membrane yielded stationary phases of a ligand concentration, binding capacity, resolution, and long term stability similar to that of the corresponding Pharmacia material. However, the membranes could be used at a higher flow rate than the columns, since they are less mass transfer limited and cause significantly less back pressure. The immobilization of high molecular weight ligands such as antibodies (immuno filtration) on the other hand, resulted in low ligand concentrations and worse antigen binding capacities whenever conventional immobilization procedures, e.g. epoxy group-based reactions, were used. In contradistinction, good results were obtained with tosyl- and tresyl activated membranes. Such membranes were successfully employed for the immobilization of monoclonal antibodies (mAb) and Concanavalin A. Concanavalin A and an anti gp 220/350 mAb were subsequently used to produce affinity filters for the isolation of a recombinant gp 220/350 Epstein-Barr virus surface antigen from culture supernatants of a Chinese hamster ovary cell line grown in protein-free medium.

Production of recombinant h-AT III with mammalian cell cultures using capillary electrophoresis for product monitoring.

The importance of mammalian cell cultures for biotechnological production processes is steadily increasing, despite the high demands of these organisms on their culture conditions. Efforts towards a more efficient bioprocess generally concentrate on maximizing the culture's life time, the cell number, and the product concentration. Here recombinant BHK 21 c13 cells are used to produce rh-AT III, an anticoagulant of high therapeutic value. The influence of the process mode (batch, repeated batch, continuous perfusion) and the process temperature (30°C vs. 37°C) on the above mentioned parameters is investigated. It is possible to increase the length of the culture from 140 h (batch) to more than 500 h (continuous perfusion culture), while concomitantly increasing the cell density from 0.72 10(6)/ml (batch) to 2.27 10(6)/ml (repeated batch) and 2.87 10(6)/ml (continuous perfusion culture). The accumulation of toxic metabolites, such as lactate, can be curtailed by reducing the bioreactor temperature from 37°C to 30°C during the later part of the exponential growth phase. Fast and reliable product monitoring became essential during process optimization. Capillary zone electrophoresis (CZE) in uncoated fused silica capillaries was studied for that purpose and compared to the standard ELISA. Under optimized conditions an AT III quantification could be done within 2 min with CZE. The detection limit was 5 µg/ml. A relative standard deviation of less than 0.9% was calculated. The detection limit could be lowered by one order of magnitude by using a two dimensional system, where an liquid chromatographic (LC) system is coupled to the CZE. Concomitantly the resolution is improved. The two-dimensional analysis required 5 min. Membrane adsorbers (MA) were used as stationary phase in the LC-system, to allow the application of high flow rates (5-10 ml/min). The correlation between the LC-CZE analysis and the standard AT III-ELISA was excellent, with r(2): 0.965. Using the assay for at line product monitoring, it is shown, that the process temperature is of no consequence for the productivity whereas the process mode strongly influences this parameter.

Fluorescein isothiocyanate-labeled protein G as an affinity ligand in affinity/immunocapillary electrophoresis with fluorescence detection.

Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10(-6) mol/L was used, h-IgG concentrations between 10(-6) and 10(-9) mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r2, of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h-IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r2 = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG1 antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG1, in human sera.

Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced fluorescence detection.

A method for the rapid and sensitive determination of immunoglobulin G (IgG) in cultivation media by an affinity assay using capillary electrophoresis is presented. For that purpose we evaluated protein A conjugated with a fluorescent dye such as fluorescein diisocyanate or dichlorotriazinyl-aminofluorescein as an affinity ligand. The ligand formed a fluorescing complex with immunoglobulin G in the sample and rapid separation from excess protein A was performed by capillary zone electrophoresis. However, only partial resolution of the zones was achieved when protein A as a whole molecule was utilized. In contrast, baseline resolution of the zones was obtained when recombinant fragments of protein A were used as affinity ligands. Immunoglobulin concentrations in the range of two orders of magnitude were determined. Due to the specificity of protein A for immunoglobulin G, analysis can be carried out even in the presence of high concentrations of other components and in cultivation media. Thus, the capillary electrophoretic affinity assay was successfully applied to monitor monoclonal antibodies in a cultivation process.