Bioanalytical Development Tools - - BioPharm International


Bioanalytical Development Tools

BioPharm International

BioArray Solutions, Ltd. announced FDA 510(k) clearance of a novel immunoassay for the simultaneous detection of six antibodies to different extractable nuclear antigens (ENA). The ENA IgG BeadChip Test System, for use on the company's Array Imaging System (AIS 400), utilizes BioArray Solutions' proprietary BeadChip format to simultaneously detect multiple analytes of interest on a tiny silicon chip holding a planar array of color encoded microparticles. The AIS 400 fully automates image acquisition and integrated analysis to rapidly generate assay results.17

Bayer HealthCare Diagnostics Division received approval from FDA for its automated HBsAg (hepatitis B surface antigen) and HBsAg Confirmatory assays.18 The fully automated tests, which are used to aid in the diagnosis and confirmation of acute or chronic hepatitis B infections, are available on the ADVIA Centaur Immunoassay System. The ADVIA Centaur HBsAg Assay is an in vitro diagnostic immunoassay for the qualitative detection of hepatitis B surface antigen (HBsAg) in human serum and plasma using the ADVIA Centaur system. The ADVIA Centaur HBsAg Confirmatory Assay is an in vitro diagnostic immunoassay for the qualitative confirmation of the presence of hepatitis B surface antigen (HBsAg) in human serum and plasma using the ADVIA Centaur system.

Procyon Biopharma Inc. entered into a licensing and distribution agreement with Medicorp Inc. granting the latter the exclusive worldwide rights to develop, manufacture and commercialize PSP94-based test kits for research purposes as well as the rights to sub-license for clinical diagnostic applications.19 PSP94 (Prostate Secretory Protein of 94 amino acids) is one of the three major proteins secreted in the seminal fluid, together with Prostate Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP). PSP94-based test kits measure the amount of free PSP94, bound PSP94 and PSP94 binding protein present in the blood, the relative ratios of which are believed to have utility in prostate cancer prognosis, diagnosis and monitoring. Recent studies to be presented at major conferences this year also indicate that PSP94-based test kits were able to predict patients suffering from a more aggressive disease. PSP94 was found to be a strong predictor of relapse post-radiotherapy as well as following radical prostatectomy.

Isoelectric Focusing

Isoelectric focusing (IEF) is a high-resolution, stand-alone technique that can be used as an analytical method or tool for protein purification. The only current book on the market, the Handbook of Isoelectric Focusing and Proteomics is a one-stop source for germane information in this discipline.20

BioPharm International ran an application article in April 2005; which discussed a case study of developing analytical methods for an API.12 One that was improved is IEF. The excerpt from this article follows:

"The first step we took in analytical support of the formulation development was to determine the protein's isoelectric point (pI) by gel IEF. The pI is the solution pH at which a protein carries no net charge. In solutions with moderate salt concentration, the solubility of most proteins is, at a minimum, near their pI. An electric field is applied to a gel matrix containing a pH gradient to separate proteins on the basis of their charge. IEF can be one of the most powerful separation methods for evaluating charge differences and can resolve a single charge difference between large protein molecules.

The first IEF test used materials purchased from Invitrogen because these were available in the laboratory. An API sample was desalted using a 5K molecular-weight-cut-off spin filter. The sample was diluted in pH 3-10 IEF sample buffer and then samples and pI markers were loaded on a pH 3-10 IEF gel. Three major, poorly resolved bands migrated between the pH 5.3 and 6.0 markers.

Because multiple bands were observed, a method for further characterization was required to ensure that formulation and process conditions were not affecting the pattern of charge variants. The IEF bands' sharpness and resolution were improved by using a larger gel on a flat-bed system. A recirculating chiller assured temperature control. Flat-bed IEF was performed using equipment and materials purchased from Amersham Biosciences. An Ampholine PAGEplate pH 3.5-9.5 gel was used, with 1M phosphoric acid as the anode buffer and 1M NaOH as the cathode buffer. After staining with Coomassie blue, the gels were placed on a white-light transilluminator and photographed with the Kodak EDAS 290 system.

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