Improved high-performance liquid chromatographic method in the analysis of adenovirus particles.

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells.

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding.

Properties of recombinant HtrA: an otitis media vaccine candidate antigen from non-typeable Haemophilus influenzae.

Non-encapsulated or non-typable Haemophilus influenzae (NTHi) is a major cause of middle ear infections in young children. HtrA has been identified as a vaccine candidate antigen from NTHi; therefore physicochemical characterization of this antigen is important for vaccine development. Recombinant NTHi HtrA has been expressed in E. coli and shown to have serine protease activity. Several mutant, recombinant HtrA proteins were expressed and purified to obtain suitable vaccine antigens lacking protease activity. Two mutants with alterations at the putative active site His91 and Ser197, designated H91A and S197A were examined by circular dichroic spectropolarimetry (CD) to evaluate secondary structure. The S197A mutant had a more random secondary structure compared to wild-type rHtrA or H91A. It is likely that improper folding of S197A accounts for its lack of immunoprotective properties in a chinchilla model of otitis media.

Methods of preparation of recombinant cytokine proteins.

Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.

Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS.

SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.

Rapid SDS gel capillary electrophoretic analysis of proteins.

Capillary sodium dodecyl sulfate (SDS) electrophoresis is a powerful and sensitive method for the qualitative and quantitative microanalysis of molecular mass for biopolymers. Usually, a separation is carried out in the long part of the capillary (20-50 cm), before the detection window, with separation times of 15-50 min. The separation of model proteins and real fractions of cell culture broth in the short part (7 cm) of the capillary, behind the window, was investigated. The analysis time could be reduced to less than 5 min. A comparison with established separation techniques is presented. Different modes of injection (by pressure and electrokinetic) were considered. The proposed method can be used for on-line monitoring of biotechnological processes.

Rapid SDS-Gel capillary electrophoresis for the analysis of recombinant NADP(+)-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification.

The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purification process were analysed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purification were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the purified FDH was determined as 44,078 Da by matrix-assisted laser desorption/ ionisation time of flight mass spectrometry.

Sodium dodecyl sulfate-polymer capillary electrophoresis for the analysis of cell culture proteins.

The application of sodium dodecyl sulfate (SDS)-polymer capillary electrophoresis (CE) to the analysis of proteins of hybridoma cell culture supernatant is demonstrated. All steps of the developed analysis are shown and discussed. Following optimization of sample preparation and concentration, as well as of the mode of injection all main protein components were separated with high resolution and selectivity. Proteolytic degradation of IgG, due to microbial contamination, was observed on SDS-gel CE and capillary zone electrophoresis (CZE). The technique was applied to monitor the cell cultivation process and concentrations of main components are demonstrated. IgG was found to increase from initially 5 mg/L to 20 mg/L after 4 days and to > 130 mg/L after 14 days of cultivation. The techniques may be applied to other biotechnological processes for the production or purification of proteins.

[Preparation of recombinant cytokines. II. Effective method for isolation, purification and renaturation of human recombinant gamma-interferon]. / Metoddy polucheniya rekombinantnykh belkov-tsitokinov. II*. 'Effektivnyl metod vydeleniya irenaturatsii rekombinantrnogo gamma-interferona cheloveka

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages. A highly active (2 x 10(7) U/mg) recombinant protein of up to 99% purity (according to SDS-PAGE and HPLC) was obtained with a 30% overall yield.

Recombinant human insulin. V. Optimization of the reversed-phase high-performance liquid chromatographic separation.

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.

Recombinant human insulin. III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin.

The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e., size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography), to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.