Validation of peptide mapping with electrospray mass spectrometry for recombinant proteins of biopharmaceutical interest and its applications as an identity test and a characterization tool.

In this paper, the steps required to validate a liquid chromatography peptide mapping method with mass spectrometric detection (LC-MS) for use as an identity test and characterization tool are presented. All aspects of peptide mapping are evaluated and optimized, including protein sample preparation (protein reduction, alkylation and enzymatic digestion), high performance liquid chromatography (HPLC) separation of the resulting peptides, and the use of a mass spectrometric detection. In addition, the validation of a single quadruple MS detector is described and the implementation of on-line electrospray ionization MS (ESI-MS) as an adjunct detector to support the investigation of peak differences is presented. Applications of peptide mapping with tandem MS using an electrospray ion-trap instrument throughout the biopharmaceutical product development cycle are discussed, including assessing protein product heterogeneity derived from post-translational modifications (e.g. multiple N- or C-termini, deamidation, oxidation and glycosylation) and protein degradation.

Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.

Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.

PURPOSE: Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis. METHODS: The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method. RESULTS: The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain. CONCLUSIONS: These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.

Association properties of betaB2- and betaA3-crystallin: ability to form dimers.

The beta-crystallins are a major constituent of the mammalian lens, where they associate into dimers, tetramers and higher order aggregates. Appropriate association of lens crystallins is important for lens transparency. To examine the associative properties of betaB2-crystallin, we have expressed mouse betaB2-crystallin using a baculovirus system. Recombinant mouse betaB2-crystallin has an estimated monomer molecular weight of 24 kDa by SDS-PAGE, appropriate immunoreactivity and appropriate secondary structure as assessed by circular dichroism analysis. The recombinant betaB2-crystallin associates into a homodimer with a weight average molecular mass of 39 kDa. The betaB2-crystallin homodimer has an estimated Kd of 5 x 10(-6) M, slightly greater than that of betaA3-crystallin, 0.8 x 10(-6) M. When recombinant betaB2-crystallin is combined with recombinant betaA3-crystallin, a heterodimer is formed within 10 min of incubation at room temperature. When equilibrium is reached in 4-6 h, approximately half of each crystallin associates into heterodimers. Subunit exchange between betaB2-crystallin and betaA3-crystallin occurs readily in the absence of any denaturing agents. Thus, rbetaA3-rbetaB2 heterodimer formation can occur under conditions similar to those found in the eye lens.

Aggregation of beta A3-crystallin is independent of the specific sequence of the domain connecting peptide.

The beta- and gamma-crystallins are structural proteins whose high concentration and close packing are important in maintaining transparency of the eye lens. The beta gamma-crystallin superfamily includes proteins with similar core structures consisting of two compact domains linked by a short connecting peptide. In gamma-crystallins, the connecting peptide folds back on itself, allowing the amino and carboxyl domains to participate in close interactions. The beta-crystallin connecting peptide is extended so that dimerization of two beta-crystallin monomers is required for similar interdomain interactions. In order to examine the role of the sequence of the connecting peptide in determining the extended beta-crystallin conformation and hence their association into dimers, we have exchanged the 10 residues of the beta A3-crystallin connecting peptide with the 9-residue connecting peptide sequence of mouse gamma B-crystallin by site-directed mutagenesis. Unaltered and modified recombinant beta A3-crystallins were expressed in a baculovirus system and purified by sequential anion exchange chromatography and gel filtration. Integrity of the recombinant crystallins was confirmed by NH2-terminal sequence analysis, immunoblots, and CD spectrometry. Reconstitution of the mutant recombinant protein with crystallins from mouse lens soluble extract resulted in aggregates of identical size distribution as normal beta A3-crystallin. We conclude that the sequence of the connecting peptide is not critical for the association of beta A3-crystallin into dimers and higher order aggregates as had been postulated.

Beta A3/A1-crystallin association: role of the N-terminal arm.

The beta- and gamma-crystallins of the lens form a protein superfamily, the beta gamma-crystallins and have highly conserved two-domain core structures. Whereas gamma-crystallins exist as monomers, the beta-crystallins associate into large aggregates. The N-terminal extensions to the core domains of beta-crystallins are postulated to be essential for their aggregation characteristics. To test this hypothesis, we compared the aggregation properties of a recombinant mouse beta A3/A1-crystallin without its N-terminal extension (r beta A3tr) to a normal recombinant mouse beta A3/A1-crystallin (r beta A3). The identity of the baculo-virus system-expressed recombinant crystallins was confirmed by gel electrophoresis, immunoblots and N-terminal sequence analysis. Circular dichroism measurements indicate that the recombinant crystallins have mostly beta-sheet conformation, similar to normal beta-crystallins. The normal r beta A3 migrates on gel filtration chromatography as a homodimer, whereas the r beta A3tr migrates mostly as a monomer. After relocating the recombinant crystallins with mouse lens soluble extract, r beta A3 migrated with the dimeric beta L2 fractions and to a lesser extent with tetrameric beta L1 fractions. The reassociated r beta A3tr migrated with the trailing edge of the beta L 2 fractions (40 kDa). These results suggest that the N-terminal arm of beta A3/A1-crystallin facilitates dimer formation and is necessary for higher-order associations.

Developmental expression of creatine kinase isozymes in mammalian lens.

Four different isoforms are thought to comprise the creatine kinase of enzymes which regulate energy metabolism through the interconversion of ADP and creatine phosphate. In addition to these well characterized isoforms, MM, MB, BB and mitochondrial creatine kinase, several uncharacterized variants with atypical electrophoretic mobility have been described. In mammalian lens, creatine kinase isoforms exhibit both a regional and developmental pattern of expression. In neonatal rat and human lens, the only isoform expressed is a variant cathodic creatine kinase. Near the time of sexual maturation (11-13 yr) there is a dramatic increase in the expression of BB creatine kinase in human lens. In rat lens, a similar pattern of isoenzyme expression is also seen near the time of sexual maturation (5-6 weeks). In the mature rat lens, in addition to the cathodic variant, there is expression of BB and, to a lesser extent, MM creatine kinase. Using a polyclonal antisera, we have localized BB creatine kinase to the cuboidal epithelial cells of the adult rat lens. This unique pattern of isoenzyme expression and developmental regulation suggests a more complex scheme for the regulation of creatine kinase gene expression than previously postulated.

The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12.

The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A. (1983) J. Biol. Chem. 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M. (1986) J. Biol. Chem. 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon. Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD. The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively. The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product. The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes. This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex.

Ribokinase from Escherichia coli K12. Nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase.

The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region. The regulatory significance of these sequence features is discussed with respect to the rbs operon. rbsK has been cloned downstream from the Serratia marcescens trp promoter on a multicopy plasmid. Cells harboring this plasmid, when grown on minimal ribose plus ampicillin, express ribokinase at the level of 2% of the soluble protein, and induction with indoleacrylic acid raises ribokinase levels another 8-fold. Ribokinase has been purified to homogeneity (216 mumol/min/mg) from a strain harboring this plasmid. Protein sequence analyses of peptides generated by cyanogen bromide cleavage and o-iodosobenzoic acid cleavage confirmed the translation initiation site and the reading frame of the DNA sequence. Amino acid compositions of native ribokinase and the C-terminal dodecapeptide agree with the predicted amino acid compositions, confirming the accuracy of the DNA sequence and the translation termination site.

The amino acid sequence of D-ribose-binding protein from Escherichia coli K12.

The amino acid sequence of the D-ribose-binding protein from Escherichia coli K12 was determined from the DNA sequence of the gene. Protein sequence analyses covering 80% of the protein were consistent with the sequence deduced from the DNA. The mature binding protein has 271 amino acid residues and shows substantial homology to D-galactose-binding protein. A signal peptide sequence of 23 or 25 residues was also deduced from the DNA sequence. It shows the characteristic features of prokaryotic signal peptides.