Array genotyping as diagnostic approach in medical genetics.

Genotyping arrays are by far the most widely used genetic tests but are not generally utilized for diagnostic purposes in a medical context. In the present study, we examined the diagnostic value of a standard genotyping array (Illumina Global Screening Array) for a range of indications. Applications included stand-alone testing for specific variants (32 variants in 10 genes), first-tier array variant screening for monogenic conditions (10 different autosomal recessive metabolic diseases), and diagnostic workup for specific conditions caused by variants in multiple genes (suspected familial breast and ovarian cancer, and hypercholesterolemia). Our analyses showed a high analytical sensitivity and specificity of array-based analyses for validated and non-validated variants, and identified pitfalls that require attention. Ethical-legal assessment highlighted the need for a software solution that allows for individual indication-based consent and the reliable exclusion of non-consented results. Cost/time assessment revealed excellent performance of diagnostic array analyses, depending on indication, proband data, and array design. We have implemented some analyses in our diagnostic portfolio, but array optimization is required for the implementation of other indications.

Sample Preparation of Secreted Mammalian Host Cell Proteins and Their Characterization by Two-Dimensional Electrophoresis and Western Blotting.

The manufacturing and purification of therapeutic recombinant proteins expressed by cultivated mammalian cells into the cell culture medium leaves the potential for contamination by host cell proteins (HCPs). Validation and quality control testing of any therapeutic protein needs to include a test to show that HCP contamination is at a minimal level. The presence of residual mammalian HCPs during purification and in the final drug product is typically determined using enzyme linked immunosorbent assay (ELISA), which is regarded as the gold standard. The complexity and heterogeneity of HCPs, which include proteins with significant differences in molecular weight (MW), isoelectric point (pI) and hydrophobicity, poses a challenge to detection and quantitation. Two-dimensional gel electrophoresis (2-DE) is one of the most powerful technologies for studying complex protein profiles and is a valuable analytical method in biologics manufacturing. In the purification process, it is very important to know the nature and composition of HCPs, and this information can be used in a rational process design in order to minimize HCPs from the product. Additionally, 2-DE in combination with western blotting can support ELISA development and quality control for the comprehensive immunochemical detection of HCPs by estimating the recognition capacity of the polyclonal serum used in those assays. Here, we present a standardized 2-DE western blotting protocol which takes into account the latest developments in sample preparation of HCPs, protein electrophoresis, protein transfer, immunostaining, and imaging.