Comprehensive Discovery and Quantitation of Protein Heterogeneity via LC-MS/MS Peptide Mapping for Clone Selection of a Therapeutic Protein.

Development of biopharmaceutical production cell lines requires efficient screening methods to select the host cell line and final production clone. This is often complicated by an incomplete understanding of the relationship between protein heterogeneity and function at early stages of product development. LC-MS/MS peptide mapping is well suited to the discovery and quantitation of protein heterogeneity; however, the intense hands-on time required to generate and analyze LC-MS/MS data typically accommodates only smaller sample sets at later stages of clone selection. Here we describe a simple approach to peptide mapping designed for large sample sets that includes higher-throughput sample preparation and automated data analysis. This approach allows for the inclusion of orthogonal protease digestions and multiple replicates of an assay control that encode an assessment of accuracy and precision into the data, significantly simplifying the identification of true-positive annotations in the LC-MS/MS results. This methodology was used to comprehensively identify and quantify glycosylation, degradation, unexpected post-translational modifications, and three types of sequence variants in a previously uncharacterized non-mAb protein therapeutic expressed in approximately 100 clones from three host cell lines. Several product quality risks were identified allowing for a more informed selection of the production clone. Moreover, the variability inherent in this unique sample set provides important structure/function information to support quality attribute identification and criticality assessments, two key components of Quality by Design.

Promoter characteristics of two cyp19 genes differentially expressed in the brain and ovary of teleost fish.

Teleost fish are characterized by exceptionally high levels of neural estrogen biosynthesis when compared with the brains of other vertebrates or to the ovaries of the same fish. Two P450arom mRNAs which derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (b>>a) and ovary (a>>b) and have a different developmental program (b>>a) and estrogen upregulation (b only). A polymerase chain reaction (PCR)-based genomic walking strategy was used to isolate the 5'-flanking regions of the goldfish (Carassius auratus) cyp19 genes. Sequence analysis of the cyp19b gene approximately 1.8 kb upstream of the transcription start site revealed a TATA box at nucleotide (nt) -30, two estrogen responsive elements (EREs; nt -351 and -211) and a consensus binding site (NBRE) for nerve growth factor inducible-B protein (NGFI-B/Nur77) at -286, which includes another ERE half-site. Also present were a sequence at nt -399 (CCCTCCT) required for neural specificity of the zebrafish GATA-2 gene, and 16 copies of an SRY/SOX binding motif. The 5'-flanking region ( approximately 1.0 kb) of the cyp19a gene had TATA (nt -48) and CAAT (nt -71) boxes, a steroidogenic factor-1 (SF-1) binding site (nt -265), eight copies of the SRY/SOX motif, and two copies of a recognition site for binding the arylhydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) heterodimer. Both genes had elements previously identified in the brain specific exon I promoter of the mouse aromatase gene. Cyp19a- and -b/luciferase constructs showed basal promoter activity in aromatase-expressing rodent pituitary (GH3) cells, but differences (a>>b) did not reflect expression in fish pituitary in vivo (b>>a), implying a lack of appropriate cell factors. Consistent with the onset of cyp19b expression in zebrafish embryos, microinjection of a green fluorescent protein (GFP) reporter plasmid into fertilized eggs revealed labeling in neural tissues at 30-48 h post-fertilization (hpf), most prominently in retinal ganglion cells (RGC) and axon-like projections to the optic tectum. Expression of a cyp19a/GFP reporter was not detectable up to 72 hpf. Tandem analysis of cyp19a and cyp19b promoters in living zebrafish embryos can be a useful approach for identifying cis-elements and cellular factors involved in the correct tissue-specific, spatial, temporal and estrogen regulated expression of aromatase genes during CNS and gonadal development.

Different catalytic properties and inhibitor responses of the goldfish brain and ovary aromatase isozymes.

The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>>A) and ovary (P450aromA>>B) and have a different developmental program (B>>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>>b) are opposite to fish pituitary (b>>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Molecular cloning of an estrogen receptor beta subtype from the goldfish, Carassius auratus.

The brain of many teleost fish species, including the goldfish Carassius auratus, expresses exceptionally high levels of cytochrome P450 aromatase (estrogen synthetase). To begin investigating the molecular and cellular targets of estrogen action in goldfish brain, a polymerase chain reaction (PCR) cloning strategy was used to isolate an estrogen receptor (ER) complementary DNA (cDNA). The 2283-bp cDNA isolated from goldfish liver encoded a protein of 568 amino acids (aa) with an estimated molecular weight of 63,539. The goldfish ER had high overall sequence identity when compared to other vertebrate ER sequences: eel (64%), human beta subtype (59%), human alpha subtype (46%), medaka (46%), and rainbow trout (47%). The highest degree of conservation was seen in the DNA-binding (94-100%) and ligand-binding (67-79%) domains. Phylogenetic analysis of the ER gene family indicated that the goldfish and eel ER are most closely related to mammalian ERbeta subtypes, whereas previously identified fish, amphibian, and avian ER forms cluster separately with mammalian ERalpha subtypes. Using the goldfish ER cDNA (here designated gfERbeta), multiple mRNA species (3.1- 8.6 kb) were detected by Northern blot analysis in goldfish liver and ovary but expression was below detection in brain. Using reverse transcription-PCR analysis, gfERbeta mRNA was detected in forebrain, mid/hindbrain, pituitary, retina, liver, ovary, and testis. Further studies are required to determine whether an additional ERalpha subtype is present in the goldfish and whether ERalpha or ERbeta forms have evolutionary precedence in vertebrates.

Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary.

Evidence to date indicates that the gene encoding cytochrome P450 aromatase (P450arom) in humans is a single member of the CYPl9 family, but multiple CYPl9 loci and isoforms have been identified in pigs. Here we report the cloning and characterization of a second member of the CYP19 family in goldfish. A search for P450arom variants was prompted by studies showing that a full-length P450arom complementary DNA (cDNA) isolated from a goldfish brain cDNA library hybridizes with a high abundance 3 kb transcript in brain RNA but fails to detect a message in ovarian RNA. A stepwise PCR cloning strategy led to isolation of a 1.9-kb cDNA, which encodes a protein of 518 amino acids and has a predicted mol wt of 58.7K. The ovary-derived P450arom (-A) shares 68-72% sequence identity with ovarian aromatases of other fish species, but only 62% identity with the homologous brain-derived P450arom (-B). Amino acid differences are distributed throughout the two goldfish P450arom forms, but presumptive functional domains are highly conserved. Both P450aromA and -B are able to aromatize [3H]androgen to [3H]estrogen when expressed in nonsteroidogenic COS cells. Southern analysis and PCR-restriction analysis of genomic DNA using discriminating probes and primers indicates that a single locus encodes the brain-derived P450aromB (CYPl9B), whereas one or two different loci encode the ovarian form (CYPl9A). Northern blot analysis revealed two P450aromA messenger RNAs (1.9 >> 3.0 kb) in ovary. Simultaneous PCR amplification with A- and B-specific primer pairs confirms that P450aromA is the only form expressed in ovaries, but shows overlapping expression of the two genes in neural tissues. Whereas P450aromB messenger RNA predominates in brain (B/A, approximately 14:1), the ratios are reversed in retina (B/A, approximately 1:25). Further studies are required to resolve the evolutionary and functional implications of multiple CYPl9 genes and P450arom isozymes in goldfish, their differential expression in brain and ovary, and whether observations can be generalized to other vertebrates.

Evolutionary and functional significance of two CYP19 genes differentially expressed in brain and ovary of goldfish.

Remarkably high levels of cytochrome P450 aromatase (P450arom) enzyme are expressed in the brains of teleost fish when compared to the ovaries of the same fish, or to the brain or ovaries of other vertebrates. Northern analysis using a full-length P450arom cDNA from a goldfish brain library indicates high accumulated levels of CYP19 mRNA in the brain but fails to detect P450arom mRNA in the ovary. The possibility of different brain and ovarian mRNA variants was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) amplification of ovarian RNA using degenerate primers led to the isolation of a 243 bp P450arom cDNA fragment with approximately 20% of nucleotide and amino acid replacements when compared to the brain cDNA. Southern analysis with sequence-specific probes indicated two distinct CYP19 loci, and this was confirmed by PCR-restriction enzyme analysis of genomic DNA. Corresponding brain- and ovary-type genomic sequences were identified in a second, diploid fish species (zebrafish), evidence that two genes are not caused per se by tetraploidy in goldfish. RT-PCR analysis of different tissues with sequence-specific primers showed high levels of the brain mRNA variant and much lower levels of the ovarian variant in neural tissues with high enzyme activity. In contrast, the ovary expressed low levels of the ovarian mRNA variant exclusively. The data imply that the expression of two CYP19 genes in goldfish is controlled by distinct regulatory mechanisms. Further studies are required to determine whether the two genes lead to functionally distinct isozymes.