Release batch assay

Remove 100 pL aliquots from the supernatant (avoid the oil ) for transmitter analysis. (Aliquots may be subdivided permitting measurements of multiple transmitters from a single sample). Treat the samples in the manner appropnate for the type of transmitter ultimately to be measured For amino acids, add TCA to 10% (v/v) for neuropeptides or catecholamines, add 3 vol of methanol 10 Store the samples at-20 C until further analysis (see Note 21). Measurements of diverse transmitters released from hippocampal synaptosomes in a typical release batch assay are shown in Fig. 3. 

Prepare stock solutions of depolarizing agents so that adding of 3 pL of the stock solution to a release batch assay tube (containing 125-150 pL) achieves the desired final concentration of depolarizing agent (i e., 40-50X stocks)... 

As long as the health authorities accept 90-110% specification limits on the drug assay, the normalization method presented above will barely suffice for batch release purposes. Since there is a general trend toward tightening the specification limits to 95-105% (this has to do with the availability of improved instrumentation and a world-wide acceptance of GMP-standards), a move toward options 1 (HPLC) and 2 (DA-UV) above is inevitable. 

The GMT in human serum reacts most rapidly with Y-glutamyl-p-nitroanilide at pH 8.2. The same activity is found in 2-amino-2-methylpropane-l 3 diol, diethanolamine, triethanolamine and tris buffers. Magnesium ions have no effect on the activity but favor the solubilization of the substrate. Bondar and Moss (54) found that free glutamate, due to elevated serum glutamate concentrations or glutamate released by substrate breakdown, increases the apparent GMT activity. They concluded that the assay should be performed in the presence of 1.0 vM/1 glutamate in order to reduce the possibility of falsely elevated results. This was not observed by others. Rowe and co-workers have indicated that certain batches of p-nitroanilide substrate contain impurities which may reduce GMT activity and increase the values ( ). Huesby and Stromme (56) confirmed the presence of such impurities and recommended pyridine extraction for substrate purification. 

Several in vitro tests are currently employed to assure drug product quality. These include purity, potency, assay, content uniformity, and dissolution specifications. For a pharmaceutical product to be consistently effective, it must meet all of its quality test criteria. When used as a QC test, the in vitro dissolution test provides information for marketing authorization. The dissolution test forms the basis for setting specifications (test, methodology, acceptance criteria) to allow batch release into the market place. Dissolution tests also provides a useful check on a number of physical characteristics, including particle size distribution, crystal form, etc., which may be influenced by the manufacturing procedure. In vitro dissolution tests and QC specifications should be based on the in vitro performance of the test batches used in in vivo studies or on suitable compendial specifications. For conventional-release products, a single-point dissolution... 

Dissolution kinetics was studied under sink conditions by placing one implant in varying volumes (usually 100 ml) of phosphate buffer pH 7.4, while agitating in a horizontally shaking water bath (50 1 rev/min) at 37 1°C. Samples were withdrawn at varying time intervals for a duration of 21 days (an estimate of the expected duration of clinical use) and the amount of tobramycin sulphate released was determined spectrophotometrically. Equal volumes of fresh medium were added to replace aliquots removed for assay and the amount of drug release was corrected for dilution. Triplicate measurements were performed for each batch of implants prepared. 

The concentration of active ingredient D1 for batch to batch is shown in Figure 13. The mean potency of all batches is 0.1 mg/5 ml above target. The control chart did not respond to tests for unnatural patterns and trends. It is noteworthy that the calculated UCL (16.7 mg/5 mL) for the 20 batches in this study exceeds the release specification for the product (15.5 to 16.5 mg/5 ml. A probability thus exists that a batch may eventually fail to meet the release criteria. Raw material purity is not a factor in the potency of an individual batch because it is taken into consideration at the time of manufacture. A possible explanation for the wide historical control limits is the assay methodology for... 

The design of the validation testing and the composition of the protocol reflect the circumstances under which the study is conducted. For retrospective validation the test may be statistical analysis of batch release data, such as assay, pH, physical appearance, residual moisture, reconstitution time, and constituted solution appearance. This retrospective process validation would be intended to demonstrate that the product is of consistent quality. A critical review of the processing conditions in a retrospective validation may consist of a test comparing actual processing conditions during lyophilization with ideal parameters. This not only shows adherence to the defined processing conditions, but also demonstrates process reproducibility. 

Potency can also be evaluated during validation. It is assumed that some number of composite assays are tested during validation. One criterion might be to generate a 100(1 - a)% confidence interval about the mean using all the potencies collected. This interval will contain the true batch potency with 100(1 - a)% confidence. This interval should be contained within the potency in-house or release limits. Enough potencies should be looked at to have sufficient power that this interval will be contained within the desired limits. 

The quality control of the final product must be carried out before release of the batch (except for the sterility and the endotoxin tests for extremely short-lived radionuclides). Consequently, all procedures must not only be very fast but also very accurate, and in all cases it is very important to have a properly established quality assurance system that might permit parametric release of the produced batches. The quality control assays that must be carried out in the radiopharmaceutical includ the following ... 

Shelf life, stability data stability-indicating assay must be provided, and evidence of product s compliance with specifications during specified shelf-life periods should be shown. Stability reports should include batch sizes and numbers, assay methods used, sampling techniques, conditions of storage and release/stability of each. 

Raw material specifications must be at the level of a recognised pharmacopoeia. However, for purposes of release of the raw material, at least the identification and assay is required and any parameter that can be prejudiced during transport. It may not be necessary to test all the pzirameters providing a vcilid certificate of analysis (CoA) is routinely available. Valid in this context meains a certificate of analysis not older than one year, reflecting the results of actucd testing cind containing the raw material batch number clearly stated and must not be confused with a certificate of compliance or specification sheet. 

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