Carolyn Roitsch

Characterization and quality control of recombinant adenovirus vectors for gene therapy.

Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.

Isolation of recombinant hirudin by preparative high-performance liquid chromatography

The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described. rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.

Protection by recombinant alpha 1-antitrypsin Ala357 Arg358 against arterial hypotension induced by factor XII fragment.

The specificity of serpin superfamily protease inhibitors such as alpha 1-antitrypsin or C1 inhibitor is determined by the amino acid residues of the inhibitor reactive center. To obtain an inhibitor that would be specific for the plasma kallikrein-kinin system enzymes, we have constructed an antitrypsin mutant having Arg at the reactive center P1 residue (position 358) and Ala at residue P2 (position 357). These modifications were made because C1 inhibitor, the major natural inhibitor of kallikrein and Factor XIIa, contains Arg at P1 and Ala at P2. In vitro, the novel inhibitor, alpha 1-antitrypsin Ala357 Arg358, was more efficient than C1 inhibitor for inhibiting kallikrein. Furthermore, Wistar rats pretreated with alpha 1-antitrypsin Ala357 Arg358 were partially protected from the circulatory collapse caused by the administration of beta-Factor XIIa.

Analysis of the primary structure and post-translational modifications of the Schistosoma mansoni antigen Smp28 by electrospray mass spectrometry.

The Schistosoma mansoni glutathione-S-transferase with an apparent molecular mass of 28 kDa, Smp28, has a blocked N-terminus which has been elucidated with the aid of the cDNA sequence combined with mass spectrometry and amino acid composition analysis of the N-terminal tryptic peptide. The blocked N-terminal tryptic peptide (m/z 695.8) contained an equimolar ratio of E, G, H, A, I and K3 upon amino acid composition analysis in agreement with its expected sequence AGEHIK, and showed a delta m = +41.7 Da compared to the predicted mass, which is consistent with the N-terminal alanine being acetylated (delta m = +42.0 Da). The mass of the complete molecule (23,744.5 +/- 3.3 Da) determined by electrospray mass spectrometry showed a further mass increase of 14 Da with respect to Smp28 containing an N-acetylated alanine. This result is consistent with one of the seven methionines being present as a methionine sulfoxide in ca. 90% of the Smp28 molecules in this preparation. Tryptic mapping of Smp28 showed five of the seven methionines to be partially oxidized by mass spectrometry. This is indicative of the ease with which this modification occurs. Two minor components were detected along with the intact molecule, corresponding to modified forms of the molecule, originating from reaction of the only cysteine residue either with itself forming a covalent dimer or with glutathione. On-line liquid chromatography-mass spectrometry has been compared with the off-line complete tryptic map of Smp28 confirming 97% of the primary structure in less than 2 h.

Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli

The membrane-associated structural protein, p18, of the human immunodeficiency virus (HIV-1), has been expressed in Escherichia coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant p18 from 16.71 of cell culture represents an overall yield of ca. 20%. Recombinant p18 was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, reversed-phase HPLC, amino acid composition and amino acid sequence analysis of the N-terminus. Edman degradation of peptides generated by trypsin or Staphylococcus aureus V8 proteolytic digestion, including the C-terminus, confirmed the amino acid sequence to be that predicted from the cDNA. A C-terminally cleaved form of recombinant p18, p18LM, was separated in the cation-exchange HPLC step and was partially characterized in parallel with the intact molecule. By Western blotting it was shown that recombinant p18 in addition to the cleaved form p18LM is recognized by a monoclonal antibody which was generated against the natural protein from HIV-1.