Stability accelerated assays

The intensive research carried out to achieve good stability and easy handling of the BL reagents allowed development of many simple, rapid, highly sensitive and specific assays, many of which are available as commercial kits. More recently application of the newest techniques of molecular biology greatly accelerated the continuous improvement of BL assay performance. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

For drug substances, a retest date is generally determined. The period up to the retest date is a period during which the material is known to adhere to its specification, for example, for assay or degradants. The retest date may be assigned on the basis of real-time stability data obtained at the recommended storage conditions or on the basis of accelerated stability data obtained under more stressful conditions. 

Continuous-monitoring methods for assay of TR-ACP activity are based on the principle introduced by Hillmann in which a-naphthoi released from its phosphate ester forms a colored product with the stabilized diazonium salt of 2-amino-5-chlorotoluene-1,5-naphthalene disulfonate (Fast Red TR). The introduction of alcohols, such as 1,5-pen-tanediol, accelerates the reaction and increases sensitivity by acting as phosphate acceptors in transfer reactions. The addition of sodium tartrate inhibits the sensitive isoenzymes (i.e., prostatic and lysosomal ACPs) if they are present in the sample. 

High throughput in-well sonication has been applied to dissolve compounds that are insoluble in DMSO in 96, 384, and 1536 well formats (Oldenburg et ah, 2005). Compounds that precipitated from DMSO stocks, due to either water uptake that reduced solubility or low instrinsic solubility that promoted crystallization, can be re-dissolved by low energ) sonication. Sonication can accelerate compound dissolution in seconds and, in some cases, drive the solution to super saturation, due to energy input and elevated temperature. This process can bring many precipitates back into solution and has no effect on compound stability. Sonication of DMSO stocks or concentrated aqueous solutions can improve HTS hit rates and enhance biological assay results. 

Analytical methods for assay of the toxicology formulations and cleaning validation are developed and validated in preparation for the first GLP studies. Release and stability testing of the toxicology test articles are performed to support the suitability of the materials through their anticipated period of use. Typically, short-term accelerated stability studies are performed on the toxicology batches for at least 3 months to cover the time from date of manufacture through the last dose. 

PiColorLock Phosphate Detection System (Innova Biosciences, Cambridge, UK) 100 pM phosphate standard. Gold reagent solution, accelerant solution, and stabilizer solution. Store at 4 °C or on ice during assay. 

Changes in physicochemical properties of the drug substance (e.g., color, crystal modification) may take place upon irradiation. Efforts should be made to observe such changes during the in vitro assay. The sample absorption spectrum should be recorded before and after irradiation surface color of solid samples should be evaluated by appropriate methods and the identity of the sample crystal modification should be confirmed when the drug is irradiated in the solid state. The humidity in the test chamber can influence the photochemical stability of certain solid samples, as demonstrated for mefloquine. The photoinduced yellowing of uncoated mefloquine tablets is accelerated by an increase in humidity. These tablets are mainly used in tropical countries and the real in-use conditions will include high relative humidity. In such cases, the influence of the humidity on the photostability must be taken into account (Tpnnesen et al., 1997). 

Practical consequences of such metastable states and associated transitions for the freeze-drying of biopharmaceuticals are self-evident. Most damaging would be the uncontrolled release of water from a hydrate, giving rise to vial-to-vial variations in water content and water release during accelerated stability assays. Other consequences include the generation, but vial-to-vial variability, of polymorph mixtures. 

---