Kinetics of pramlintide degradation in aqueous solution as a function of temperature and pH.

The stability of the 37-amino acid peptide pramlintide, in aqueous solution, was studied as a function of pH and temperature. Samples of pramlintide formulated as a parenteral product were exposed to elevated temperatures and to realistic storage conditions for as long as 30 months. Pramlintide degradation was monitored by three high-performance liquid chromatography (HPLC) methods: a reversed-phase (RP-HPLC) and a strong-cation exchange (SCX-HPLC) method for percentage purity determination by area normalization, plus a second RP-HPLC method for potency determination versus external standards. The pH-rate profile for pramlintide shows increasing degradation rate constants with increasing pH over the range pH = 3.5 to 5.0. The Arrhenius expression for pramlintide degradation at pH = 4.0 over the temperature range 5 degrees C to 50 degrees C is ln(k(0))= 37.39-21.900/RT, where k(0) is the zero-order rate constant (in %/mo) for pramlintide degradation. The pramlintide parenteral product formulated at pH = 4.0 is extremely stable, with percentage purity and percentage potency loss of only approximately 2% over 30 months at 5 degrees C. The formulated pramlintide drug product has acceptable shelf life for long-term storage at 5 degrees C and up to a 30-day patient use when stored at ambient temperature.

Pramlintide injection drug product robustness studies.

The article examines the effects of temperature excursions and actual dose withdrawal on the quality of pramlintide injection, a multidose liquid parenteral formulation. Studies were designed to demonstrate product robustness under conditions that may occur during patient use. Pramlintide %Purity was determined by two high-performance liquid chromatography (HPLC) methods, a reversed-phase (RP-HPLC) and a strong-cation exchange (SCX-HPLC) method. A second RP-HPLC method was used to determine pramlintide potency and the concentration of the m-cresol preservative. Antimicrobial preservative effectiveness testing was per USP and European Pharmacopeia (Ph. Eur.). Short-term stability studies were undertaken to probe the effects of the following conditions: 5 degrees C to 40 degrees C and 5 degrees C to -20 degrees C temperature cycling over 10 days; once daily or four-times daily dose withdrawal over 12 or 42 days; and combined 30 degrees C storage and four-times daily dose withdrawal over 42 days. In all cases, pramlintide %Purity and potency values remained essentially unchanged or unchanged relative to controls. Similarly, product appearance, and m-cresol concentration and preservative effectiveness were not significantly affected by the stress conditions used in the 5 studies. Pramlintide injection drug product is extremely robust to challenging stress conditions that may occur during patient use of this multidose product for chronic administration.

Effects of fermentation on product consistency.

A variety of different fermentation processes has been successfully employed to produce consistent protein-based biopharmaceuticals from genetically engineered animal cells. Chinese hamster ovary (CHO) cells were genetically modified to produce recombinant human soluble CD4, tissue plasminogen activator (tPA) or erythropoietin (EPO). Soluble CD4 was collected from extended perfused fermentations of several months' duration, during which some quantitative loss of DNA copy level, mRNA expression level, and fermentation titer were observed. In one extended run, a novel contaminant appeared in intermediates purified from later harvests. However, in all cases, the final soluble CD4 product was consistent in terms of purity and potency. Evaluation of genetic stability for tPA examined both biological traits at the cellular level as well as potency, purity and structure of product derived from cells at various levels of in vitro age; no significant cell age effects were observed. Similarly, evaluation of the EPO product showed that genetically-determined and process-determined traits such as potency, tryptic peptide mapping, and sialylation were consistent from lot to lot. These data exemplified how process design, process validation, and in-process and quality control assays can be used effectively to ensure the consistency of recombinant products derived from cell culture fermentations.

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins.

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.

Protection of cattle from bovine herpesvirus type I (BHV-1) infection by immunization with individual viral glycoproteins.

The major glycoproteins gI, gIII, and gIV of bovine herpesvirus-1 (BHV-1) were found to induce high levels of antibody in cattle which could neutralize virus and participate in antibody-dependent cell cytotoxicity of BHV-1-infected cells. Immunized animals were fully protected from disease, using a BHV-1/Pasteurella haemolytica aerosol challenge model but not from infection with the virus. Thus, virus could still replicate in the nasal passages of immunized animals, although to a lesser extent than in placebo-treated animals or animals immunized with a commercial killed whole virus vaccine. Systemic spread of the virus in immunized animals did not appear to occur since there was not a dramatic alteration of leukocyte function following challenge. These results suggest that any one of the three major BHV-1 glycoproteins may be useful as a subunit vaccine either individually or in combination.

Trypanosoma congolense: structure and molecular organization of the surface glycoproteins of two early bloodstream variants.

The complete primary structures of two variant specific glycoproteins (VSGs) of the nannomonad Trypanosoma (N.) congolense are presented. These coat proteins subserve the function of antigenic variation. The secondary structure potentials of both VSGs have been calculated. The amino acid sequences and secondary structure potentials of these VSGs have been compared with the primary structures and secondary structure potentials of several Trypanosoma brucei complex VSGs. In homologous regions, the T. brucei complex VSGs show a pattern of sharply contrasting secondary structure potentials. It has been suggested previously that this pattern gives rise to different folding structures in different members of this polygene protein family. Thus, different short regions of the polypeptide sequence are exposed as antigenic "caps" on the solvent-exposed surface of intact trypanosomes. A sharply contrasting secondary structure potential pattern is also found in regions of the two T. congolense VSGs. However, there is little homology of primary structure between each of the two T. congolense VSGs and any member of the T. brucei complex VSG polygene family whose primary structure has been determined.

Microscale elucidation of an N-linked glycosylation site by comparative high-performance liquid chromatographic peptide mapping.

The identification of specific sites of post-translational modifications of polypeptides is an important component in understanding protein structure-function relationships. This report describes the application of specific enzymatic methods of N-linked oligosaccharide removal (endo H and endo F) to the rapid microscale identification of specific sites of N-linked glycosylation. Following deglycosylation the protein is subjected to proteolytic digestion and comparative high-performance liquid chromatographic peptide mapping with an unmodified protein digest. Peptides from which oligosaccharide(s) have been removed exhibit a measurable increase in retention time on reversed-phase high-performance liquid chromatography and can be clearly identified. Once identified, the peptide can be subjected to direct protein microsequence analysis to elucidate the specific site of glycosylation. As illustrated here, these methods are compatible with microscale protein purification by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by electroelution and are applicable to the 100-pmol range.

Streptococcal pyrogenic exotoxin type A (scarlet fever toxin) is related to Staphylococcus aureus enterotoxin B.

The nucleotide sequence of the gene encoding group A streptococcal pyrogenic exotoxin type A (SPE A) was determined by the dideoxy chain termination method. The first 30 residues of the translation product represented a hydrophobic signal peptide. The mature protein was 220 amino acids in length and had a molecular weight of 25,805. It has significant protein sequence homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library.

High-level expression in Escherichia coli of biologically active bovine growth hormone.

High-level synthesis of bovine growth hormone (bGH) in Escherichia coli was achieved by maximizing gene transcription and optimizing the translational efficiency of bGH mRNA. Nearly all of the recombinant hormone was found in the pellet fraction after bacterial cell lysis. This property allowed the purification of bGH nearly to homogeneity. Protein sequence analysis indicated that greater than 93% of the purified hormone had the amino-terminal methionine residue removed by E. coli, yielding mature bGH. In a hypophysectomized rat assay system, purified bacterial-produced bGH demonstrated growth-promoting activity equivalent to that of pituitary-derived bovine growth hormone.

Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins.

A vector system has been designed for obtaining high yields of polypeptides synthesized in Escherichia coli. Multiple copies of a synthetic gene encoding the neuropeptide substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) have been linked and fused to the lacZ gene. Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide (CNBr). The isolated multimeric SP fusion protein was converted to monomers of SP analog, each containing a carboxyl-terminal homoserine lactone (Hse-lactone) residue (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse-lactone), upon treatment with CNBr in formic acid. The Hse-lactone moiety was subjected to chemical modifications to produce an SP Hse amide. This method permits synthesis of peptide amide analogs and other peptide derivatives by combining recombinant DNA techniques and chemical methods.

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

A hydrophobic tryptic peptide from bovine white matter proteolipid.

A hydrophobic, chloroform-soluble tryptic peptide with a molecular weight of approximately 4000 has been purified from the bovine white matter proteolipid protein. Its primary structure was obtained by a combination of solid-phase Edman degradation and mass spectrometry. A major part of the tryptic peptide appears to be inaccessible to the action of proteolytic enzymes. The peptide spans the three cyanogen bromide peptides located by Jollès et al. (Biochem. Biophys. Res. Commun. (1979) 87, 619--626) at the COOH-terminal region of the intact protein. Secondary structure calculations for this region indicate a segregation into discrete domains, with most of the tryptic peptide corresponding to a highly ordered, hydrophobic domain; an equal probability for alpha-helical or beta-structure is predicted for this region.

Primary structure of the hydrophobic plant protein crambin.

Crambin, a hydrophobic plant seed protein, consists of a single chain of 46 amino acids with a calculated molecular weight of 4720. The primary structure was determined by using solid-phase sequencing techniques and was confirmed through X-ray crystallographic analysis of the protein at 1.5-A resolution [Hendrickson, W. A., & Teeter, M. M. (1981) Nature (London) 290, 107-112]. High-performance liquid chromatographic separation of the proteolytic fragments from crambin led to the identification of two sites of microheterogeneity. The three disulfide bonds were located at positions 3-40, 4-32, and 16-26 from the crystallographic data. Comparison of the primary structure with known sequences revealed that crambin is homologous with the plant toxins purothionin and viscotoxin. Methods to estimate protein secondary structure were applied and found to predict all of crambin's structure except its amphiphilic helix.

The amino acid sequence of elongation factor Tu of Escherichia coli. The large cyanogen bromide peptides.

The amino acid sequences of three large cyanogen bromide peptides, which comprise 289 of the 393 amino acids in Escherichia coli elongation factor Tu, have been determined. The peptides were digested with trypsin, Staphylococcus aureus protease, and chymotrypsin to give overlapping subfragments, which were separated by high performance liquid chromatography. Peptides were sequenced by solid-phase Edman degradation. Sequence analysis of these peptides completes the sequence of elongation factor Tu described in the companion article (Laursen, R. A., L'Italien, J. J., Nagarkatti, S. N., and Miller, D. L. (1981) J. Biol. Chem. 256, 8102-8109).

The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.

The complete amino acid sequence of elongation factor Tu of Escherichia coli has been established by sequencing overlapping cyanogen bromide and tryptic peptides. Sequence analysis of peptides was done primarily by solid-phase Edman degradation. Elongation factor Tu is a single chain polypeptide composed of 393 amino acids (Mr = 43,225). Its NH2 terminus is blocked with an acetyl group, as determined by mass spectroscopy, and lysine 56 is partially methylated. The cysteine residues associated with aminoacyl tRNA and guanosine nucleotide binding are located at positions 81 and 137, respectively. Although elongation factor Tu is coded for by two genes, the only site of microheterogeneity found was at the carboxyl terminus (residue 393), which is either glycine or serine. Comparison of the first 140 amino acids of elongation factor Tu and of elongation factor G shows a strong (31%) sequence homology. In addition, secondary structure calculations predict remarkable conformational similarities between the two proteins. The NH2-terminal region of elongation factor Tu appears to be composed of two beta-sheet domains connected by an exposed, alpha-helical bridge, which includes a 14-amino acid segment released by limited treatment with trypsin. Structural features of the aminoacyl-tRNA binding site are discussed in the light of sequence and other chemical and biochemical data.

Primary structure of elongation factor Tu from Escherichia coli.

The amino acid sequence of elongation factor Tu (EF-Tu) from Escherichia coli has been determined. EF-Tu is a single-chain polypeptide composed of 393 amino acids (Mr 43,225 for the species bearing COOH-terminal serine). The NH2-terminal serine is acetylated, and lysine-56 is partially methylated. The sites of facile tryptic cleavage are at arginines 44 and 58 and at lysine-263. The cysteinyl residues associated with aminoacyl-tRNA and guanosine nucleotide binding activities are residues 81 and 137, respectively. The COOH-terminal amino acid is heterogenous in that analyses of the COOH-terminal peptides isolated from different EF-Tu preparations gave position 393 as glycine and serine in ratios (Gly/Ser) ranging from about 0.7 to 3.