Product potency

Any biopharmaceutical must obviously conform to final product potency specifications. Such specifications are usually expressed in terms of units of activity per vial of product (or per thera- 

Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis Two-dimensional electrophoresis Capillary electrophoresis Peptide mapping HPLC (mainly RP-HPLC) 

Bioassays represent the most relevant potency-determining assay, as they directly assess the biological activity of the biopharmaceutical. Bioassay involves applying a known quantity of the substance to be assayed to a biological system that responds in some way to this applied stimulus. The response is measured quantitatively, allowing an activity value to be assigned to the substance being assayed. 

All bioassays are comparative in nature, requiring parallel assay of a standard preparation against which the sample will be compared. Internationally accepted standard preparations of most biopharmaceuticals are available from organizations such as the World Health Organization (WHO) or the United States Pharmacopeia. 

Bioassays of related substances can be quite similar in design. Specific growth factors, for example, stimulate the accelerated growth of specific animal cell lines. Relevant bioassays can be undertaken by incubation of the growth-factor-containing sample with a culture of the relevant sensitive cells and radiolabelled nucleotide precursors. After an appropriate time period, the level of radioactivity incorporated into the DNA of the cells is measured. This is a measure of the bioactivity of the growth factor. 

Any biopharmaceutical must obviously conform to final product potency specifications. Such specifications are usually expressed in terms of units of activity per vial of product (or per therapeutic dose, or per mg of product). A number of different approaches may be undertaken to determine product potency. Each exhibits certain advantages and disadvantages. 

An example of a straightforward bioassay is the traditional assay method for antibiotics. Bioassays for modern biopharmaceuticals are generally more complex. The biological system used can be whole animals, specific organs or tissue types, or individual mammalian cells in culture. 

---

Standardization and Testing". RequHemeats are geaerally specified within Hceases Hi the United States, and include a variety of Hi-process tests to assess purity, safety, and potency of the iadividual components and potency and safety of the final product. Potency is standardized by determining the size of the conjugate and the quantitative amount of saccharide that is bound to the carrier protein. General safety and immunogenicity is assessed Hi animals. 

After the product has been filled (and sealed) in its final product container. QC personnel then remove representative samples of the product and carry out tests to ensure conformance to final product specification. The most important specifications will relate to product potency, sterility and final volume fill, as well as the absence of endotoxin or other potentially toxic substances. Detection and quantification of excipients added will also be undertaken. Product analysis is considered in Chapter 7. 

Although bioassays directly assess product potency (i.e. activity), they suffer from a number of drawbacks, including ... 

Regulatory specifications may differ from product release specifications. Regulatory specifications assure acceptable product potency until the labeled expiration date or shelf-life. In general, in-house release specifications are tighter than regulatory specifications. 

The freeze-dried product contains the freeze-dried biological material, residuals of the manufacture of the product such as buffer, and any excipient such as lactose, sucrose, mannitol, sodium chloride, or sorbitol that has been added to the product to optimize the freeze-drying process and protect product potency. The types of water present in the freeze-dried product cake may be different for each product depending on the residuals of manufacture and the excipients present the water could be surface water, bound water, or trapped water. 

Evidence continues to accumulate in the literature attesting to loss in product potency or activity associated with elevated moisture content in freeze-dried products. 

Before the performance of method transfer activities involving protocols and acceptance criteria, it was customary for a receiving laboratory to repeat some or all of the validation experiments. This laboratory was thereby deemed to be qualified as described above. The choice of validation parameter(s) depends highly on the type of method being transferred. For example, content uniformity assays to determine consistency of product potency depend heavily on the method and system precision. As a second example, a determination of trace impurities in an API could not be reproduced between two sites if their instruments did not yield similar limits of detection and limits of quantitation. A detailed discussion on the rational choice of validation parameters that would need to be repeated by the receiving laboratory is beyond the scope of this chapter. The reader is referred to the method validation chapter by Crowther et al. for additional information on this subject. 

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

Pershing, L.K., Reilly, C.A., Corlett, J.L., and Crouch, D.J., Assessment of pepper spray product potency in Asian and Caucasian forearm skin using transepidermal water loss, skin temperature and reflectance colorimetry, J. Appl Toxicol, 26, 88-97, 2006. 

In addition, both products were subjected to size-exclusion chromatography to examine for the presence and extent of protein aggregation, a process that can result in loss of product potency. FVIII aggregates were not detected in either of the two samples. 

Apart from pure quahty control topics, reliable analytical methods are important to assess the pharmacokinetic profile of drags and their metabohtes, and often a correlation of pharmacokinetic and pharmacodynamic parameters helps to predict therapeutic effects. In the case of biopharmaceuticals, a new set of analytical methods had to be developed due to the different nature of these products. Potency assays were rarely used in conventional small-molecule analysis, but have gained special attention for biopharmaceuticals. A series of assays was estabhshed which are useful both in quahty control and in in vivo analytics. A lot of attention is paid to... 

Production controls should be implemented that account for the solubility characteristics of the drug substance inadequate controls can adversely affect product potency, efficacy, and safety. For example, in one instance, residual water remaining in the manufacturing vessel, used to produce an ophthalmic ointment, resulted in partial solubilization and subsequent recrystallization of the drug substance the substance recrystallized in a larger particle... 

Product Potency Tests. Two assays were developed that measure the potency of the FGF-4 protein. In the first case, a one-step growth promotion assay is conducted on normal, human retinal pigment epithelial cells (ARPE-19). The assay measures metabolic activity (Alamar blue dye metabolism) after infection of ARPE-19 cells with a serial dilution of the drug substance (Ad5FGF-4). The increase in metabolic activity correlated with FGF-4 protein production (determined by an FGF-4 ELISA), increased de novo DNA synthesis (measured by BrdU incorporation), and an increase in cell number. 

Topicals are intended for use on human skin. Therefore, they should be tested on a suitable surface. Many studies on topicals have used hard surface carriers, or even suspension tests, and extrapolated the results on product potency to human skin. We do not believe this to be a valid approach because skin may present a greater challenge than a hard inanimate surface [26] and a much greater challenge than suspension tests. Mucous membranes may present an even greater challenge. 

---