Drug potency assays

VALIDATION PRACTICES Table 2.1. Guidelines for Drug Potency Assay... 

Besides potency assessment of the purified drug, potency assays are also applied for quantification of the protein activity in biological matrices from animals/humans. Since the concentration of the therapeutic protein in the biological sample is generally low (pico- to nanomolar), standard... 

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... 

The curve is identical to the first but the axes are labelled differently with percentage of maximum response on the y axis. This graph will have been produced from a functional assay in the laboratory on a single subject and is concerned with drug potency. Demonstrate that the EC50 is as shown. 

Analytical methods are important not only in the development and manufacture of commercial biopharmaceutical drugs, they also play a vital role in the whole drug development life cycle. Drug discovery and preclinical research require development and application of analytical methodologies to support identification, quantitation, and characterization of lead molecules. It is difficult to perform a comparative potency assay on lead molecules if one does not know how much of each is going into the assay or how pure the molecule is. Analytical methods are typically developed, qualified, and validated in step with the clinical... 

For the determination of potency assay of a drug substance or a drug product, the usual range of linearity should be +20% of the target or nominal concentration. For the determination of content uniformity, it should be 30% of the target or nominal concentration. Figure 3 illustrates the linearity of a sample set of data. 

An example of the minimum requirement for potency assay of the drug substance and drug product is tabulated in Table 4. Note that the postponement of intermediate precision is aligned with previous discussion that the use of early phase analytical method resides mainly in one laboratory and is used only by a very limited number of analysts. Each individual company s phased method validation procedures and processes will vary, but the overall philosophy is the same. The extent of and expectations from early phase method validation are lower than the requirements in the later stages of development. The validation exercise becomes larger and more detailed and collects a larger body of data to ensure that the method is robust and appropriate for use at the commercial site. 

Analytical Development of API and Drug Products. Early methods to support synthetic and formulation developments are often developed in the form of potency assay, impurities/related substance assay, dissolution, Karl Fischer, identity, chiral method, and content uniformity. These analytical methods are developed and validated in a fast and timely manner to support all phase II studies. 

ICH Q2A [1] proposed the guidelines shown in Table 2.1 for the validation of a potency assay for a drug substance or drug product. 

Determination of drug potency in the presence of other emulsion components may require development of special analytical techniques for example, oils may interfere with standard reverse phase HPLC assays, requiring extraction techniques or the development of normal phase assays. Similarly, bioassays may give erroneous results when the drug is presented in an emulsion form. Hence, adequate controls and/or extraction techniques must be developed to give reliable values from the bioassay. 

Figure 8 is a plot of mean assay results for active ingredient Bl. Drug potency (200 mg per tablet) is measured in duplicate from samples obtained by grinding a composite of 20 randomly selected tablets. Figure 8 is also influenced by the variability of the purity of the raw material, which ranged from 97.6-99.5%. 

A table of values can be generated using a quasi-simulation from the above formula. (See Table 1.) If one uses the specification of 90.0-110.0% which are frequently used for potency assays of drugs products, and a method intermediate precision (as percentage ofRSD) of2.0 and 2.5%, the values obtained from Table 1 are five and seven samples, respectively. Coincidentally, these values approximate those mentioned in the U.S. v. Barr decision. It should be mentioned that this method of calculating the number of retests may not work well for some tests, such as LAL bioassays, which produce colony counts that are not normally distributed, but skewed. 

Potency of drug candidates is typically assessed in vitro. An appropriate potency assay must be biologically relevant to the clinical indication for which the drug is targeted. For example, the potency of a growth factor whose action is believed to induce proliferation of epithelial cells in vivo would need to be assessed by an in vitro epithelial cell-based proliferation assay. 

Attempts to incorporate the side chain into ring structures also led to compounds with attenuated activity. For example, in drug discrimination assays using rats trained to recognize the effect of (+)-amphetamine (10), compounds (23)and (24) either failed to produce amphetamine like effects, or had much lower potency (150, 151). When n = 3, the compound lacked any amphetamine-like action. 

Most of these studies require analysis of the formulation in terms of the potency assay of the drug substance and other critical excipients. Consequently, an analytical method such as HPLC is required. 

TABLE 2 Percent Label Claim Data for a Drug Product Potency Assay Transferred to Four Sites Simultaneously... 

Since most drugs are chromophoric (exhibit UV absorbances), HPLC with UV detection is commonly used in assays for drug potency of drug substances and products. Typical method attributes, requirements, specification limits, and acceptance criteria are listed Tables 6.2 and 6.3. Assay methodologies of many common drug substances and products are published in the United States Pharmacopeia (USP) or European Pharmacopeia (EP) monographs. Assays for potency are performed for product release and stability evaluation. 

Quasi-quantitative assays traditionally include measures of enzymatic or ligandbinding activity, as in flow cytometry and anti-drug antibody assays [9]. One of the common characteristics of these assays is the lack of a true reference standard, where reference standards are poorly characterized, do not completely represent native protein, or differ from native proteins in terms of potency or immunoreactivity. As stated above, if the analytical response is continuous across the range in question, the analytical results can be expressed in terms of a characteristic of known test samples. The following is one example an ELISA qualified as a quasi-quantitative assay because it could not be validated as a relative quantitative assay. 

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