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.

PDGF-D, a new protease-activated growth factor.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.

Integrated YAC contig containing the 3p14.2 hereditary renal carcinoma 3;8 translocation breakpoint and the fragile site FRA3B.

An extended YAC contig has been developed for the 3p14 region containing the hereditary renal carcinoma 3;8 translocation breakpoint and the 3p14.2 fragile site FRA3B. This region of chromosome 3 has been implicated by chromosomal translocation, deletion, and loss of heterozygosity in the pathogenesis of several malignant diseases. The contig allows accurate positioning of candidate genes, polymorphic markers, and other 3p rearrangements within this region. The contig, spanning approximately 6 Mb of DNA, contains 51 YACs identified by 27 markers, including a subset of CA repeats located in the 3p14.1-14.2 interval. The order of CA microsatellites, derived from marker content of the YACs, is in agreement with the order previously determined by genetic linkage studies. We find that the protein-tyrosine phosphatase gamma gene, PTPRG, is located minimally 1 Mb proximal to the t(3;8) breakpoint. The more proximal 3p homozygous deletion in the small-cell lung cancer cell line, U2020, is more than 5 Mb from the site of the 3;8 translocation. This integrated physical and genetic map provides a framework for further investigations of malignant diseases associated with proximal 3p loss. In addition, the positioning of separate 3p14.2 aphidicolin-induced breakpoints suggests that FRA3B may represent a region rather than a single site.

Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint.

The chromosome (p14.2;q24.1) translocation t(3;8) has been associated with hereditary renal cancer in one family. Based on cytogenetic analyses and loss-of-heterozygosity experiments, the 3p14 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development. The 3p14.2 region also contains FRA3B, the most sensitive fragile site induced by aphidicolin. A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was positioned between the t(3;8) breakpoint and an aphidicolin-induced 3p14 breakpoint. A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphidicolin-induced breakpoint on its telomeric side. A subsequent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint. A lambda sublibrary allowed isolation of clones spanning the rearrangement. Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library. We have identified a gene, referred to as HRCA1 (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint. On the basis of its chromosomal position, HRCA1 may be a candidate tumor suppressor gene.