Michael Mccaman

Proteomic Analysis for the Assessment of Different Lots of Fetal Bovine Serum as a Raw Material for Cell Culture. Part IV. Application of Proteomics to the Manufacture of Biological Drugs

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE‐19) using three different lots of fetal bovine serum (FBS‐Ia, FBS‐Ib, and FBS‐II). We found that the growth rate of the culture was significantly and consistently higher in the FBS‐II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional‐related and cell‐spreading‐related proteins, decreased over time.

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

Enzymatic properties and processing of bovine prochymosin synthesized in Escherichia coli

Abstract Active chymosin has been produced from calf prochymosin synthesized in E. coli . A preprochymosin cDNA clone was used to construct a plasmid, pWHA43, which expresses methionyl-prochymosin. This zymogen protein is synthesized in the bacteria in a stable but insoluble form localized in cytoplasmic inclusion bodies. Partially purified and solubilized prochymosin from E. coli can be processed by the same apparent mechanism as authentic calf prochymosin upon activation with acid. The chymosin thus derived from E. coli showed the same substrate specificity and the same kinetics of activation as that of chymosin derived from purified calf stomach prochymosin. These results also demonstrate that the bovine prochymosin synthesized in E. coli can be reconstituted and activated to a form having the functional properties necessary for an industrial milk coagulant to be used in cheese manufacturing.

Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

ABSTRACT The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 × 1012 vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39-kDa surface protein.

A tandemly linked set of four open reading frames (ORFs), identified as vspA-D (variable surface protein) had been identified from previous cloning and sequencing of clones from a genomic library constructed from Brachyspira hyodysenteriae strain B204. The predicted translation products of these closely related genes were homologous to (but not identical with) a characterized 39-kDa surface-exposed membrane protein from this animal pathogen. Additional screening of the genomic library has been performed to retrieve what are believed to be additional vsp genes including the one expected to encode this 39-kDa protein. Four new vsp genes have been identified and found to be associated in a second set of four tandemly linked alleles. This new gene cluster of 7481 nucleotides is not adjacent to the original vspA-D gene cluster described but does appear to have arisen from a gene (region) duplication event. The new vsp genes (identified as vspE-H) are oriented parallel to one another and appear to have a set of similar but distinct regulatory elements that may control separate expression of their ORFs. The four adjacent ORFs are of similar size (361-390 codons) and share from 83% to 90% identity in their amino acid sequence. The organization and homologies of these highly conserved multiple gene copies are discussed.

Sequence characterization of two new members of a multi-gene family in Serpulina hyodysenteriae (B204) with homology to a 39 kDa surface exposed protein: vspC and D

Previous cloning and sequencing of clones from a genomic library constructed from Serpulina hyodysenteriae B204 had identified a tandem pair of open reading frames, identified as vspA and vspB (variable surface protein) expected to encode proteins with homology to ( but not identical with) a 39 kDa surface exposed membrane protein from this animal pathogen. Additional screening of the genomic library was performed to retrieve the remainder of the vspB gene using new oligonucleotide probes based upon the cloned gene sequences. Not only was this goal met but we also discovered two more adjacent and related vsp genes (vspC and vspD) and have completely sequenced them. They are all in a parallel orientation and appear to have a set of similar but distinct regulatory elements that may control separate expression of their open reading frames (ORFs). Thus, there are four contiguous vsp genes which are predicted to encode a family of structurally conserved proteins. The four adjacent open reading frames (ORFs) are of similar size (384-389 codons) and share from 83% to 90% identity in their amino acid sequence. Preliminary data suggests there may be yet another homologous gene copy in a distal location of S. hyodysenteriae that faithfully encodes the 39 kDa surface protein. The organization and homologies of these highly conserved multiple gene copies are discussed.

Analytical Assays to Characterise Adenoviral Vectors and Their Applications

Recombinant adenovirus preparations are used for gene delivery in a number of clinical gene therapy approaches. Adenovirus offers short term (transient) gene expression without integration into the host cell genome1. The adenovirus is a complex biological system containing several different structural proteins and a linear double stranded DNA molecule held together by non-covalent interactions2. The classical assays used to characterise the adenovirus preparations include infectivity analysis (typically a plaque assay format3), SDS-PAGE analysis of the adenoviral proteins2,4,5 and the estimation of virus concentration by lysing the virus in SDS and measuring absorption at 260 nm in the lysate2.