Testing the In-Vitro Product Performance of Nanomaterial-Based Drug Products: View of the USP Expert Panel

Today, a wide variety of nanomaterial-based drug products enter the US market, creating the need for reliable standards and technologies to measure their performance in-vitro. A growing number of new performance assays are evaluated for testing the drug release from nanomaterials. On the one hand, they include real time separation methods such as dialysis, fiber optical systems, and flow-separation techniques. On the other hand, sample-and-separate methods such as centrifugation, filtration, and solid-phase extraction are commonly used. In our evaluation of the existing practices, we provide guidance in method development and validation of release assays. Also, we discuss requirements for standardization and documentation of release data. Furthermore, we highlight the knowledge gaps and challenges associated with drug release testing of nanomaterial-based drug products. dx.doi.org/10.14227/DT290122P6 Reprinted with permission. © 2021 The United States Pharmacopeial Convention. All rights reserved. Correspondence should be addressed to: Kahkashan Zaidi, Senior Principal Scientist, United States Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852-1790; email: kxz@usp.org. INTRODUCTION Over the years, a wide variety of nanomaterialbased drug products have entered the global healthcare market (1, 2). A recent USP chapter, Drug Products Containing Nanomaterials <1153>, provides clarification on terminology including liposomes, nanoparticles, nanocrystals, micelles, nanobubbles, nanofibers, nanotubes, nanoemulsions, and dendrimers. These novel dosage forms are characterized by exceptionally small dimensions and specific properties that make them more difficult to evaluate using conventional methodologies. In the following article, recent trends and developments in the area of in-vitro performance testing of nanomaterialbased drug products will be discussed. For some of these dosage forms such as semisolids or inhalation products, the sample collection plays an important role. These issues will be discussed in more detail by other Stimuli articles and are beyond the scope of the present work. CURRENT USP FRAMEWORK Currently, <1153> summarizes dosage forms that exhibit specific features related to the use of nanotechnology and provides the terminology to be used in the context of the USP framework. To a certain extent, the versatility of drug delivery concepts is reflected by the performance parameters that have been discussed for 7 FEBRUARY 2022 www.dissolutiontech.com nanomaterials. These performance parameters include the drug release, the physical stability or "dispersibility" of colloids, the ability of the carrier to protect the drug from degradation, as well as the release of compounds into specific compartments known to influence the biodistribution, such as the plasma proteins (3) or lipids (4). This versatility is reflected in different sections of the USP as well. For example, Gene Therapy Products <1047> recognizes the use of liposomes or lipid complexes to enhance cell penetration of deoxyribonucleic acid (DNA) molecules as well as the impact of complexation on the shelf life of the drug product. Other than the dissolution of the drug, the protective effect of the material on the compound essentially contributes to the performance of the drug product. Conversely, In Vitro Release Test Methods for Parenteral Drug Preparations <1001> emphasizes the drug release and provides selected information on some of the most common methods applied to the testing of nanosuspensions and liposomes. In the future, the interplay between the release and stabilization of drugs will play a more dominant role. Therefore, the next generation of release media should simulate both, the microenvironment relevant for dissolution and release of the drug as well as the multiple influences on drug and formulation stability (5). In this article, we focus on separation and isolation methods used in the detection of the free and the particlebound fractions of the drug. Also, we provide some recommendations for the development and validation of the release assay. REAL-TIME SEPARATION METHODS Real-time separation methods apply continuous separation or detection to identify the free fraction and the encapsulated fraction of the drug. In the following sections, these methods will be discussed in more detail. Selected assays and protocols described in the current literature are provided in Table 1. They follow certain standards but represent only a small fraction of those available in the literature. Dialysis Methods Background Today, a variety of dialysis methods are considered to measure the drug release from nanomaterial-based drug products. They utilize the inherent barrier properties of the dialysis membrane to separate colloids from drug molecules and proteins (Fig. 1). Commonly, the dosage form is filled into the donor chamber. The donor compartment is separated from the acceptor compartment by a dialysis membrane. After injection of the sample into the donor chamber, the concentration gradient between both compartments drives the exchange of molecules. The free drug is quantified from the acceptor compartment. Reverse dialysis refers to a process where a compartment that is larger in volume is used as the donor compartment. For both setups, there are two kinetic processes involved in the drug release profile: the release of the drug from the carrier and the permeation of the drug through the dialysis membrane. The rate of the membrane flux (J) is described by Fick's law of diffusion and depends on the diffusion coefficient (D), the membrane surface area (A), the concentration gradient between the donor and the acceptor compartment (dc), and the thickness of the diffusion layer (h). J = (D × A × dc) / h It can be summarized in the membrane permeation rate constant (kM). The medium exchange between both compartments is affected by membrane permeability as well (18). Conventional approaches often use dialysis cassettes or tubes with a well-defined surface area (19). They come with several technical challenges such as the agglomeration of the nanomaterial in the donor chamber. This can lead to the formation of a diffusion layer and, in consequence, to prolonged membrane transport. In this context, special attention should be paid to the viscosity of the drug product in the donor and the acceptor chambers (20). For validation purposes, the permeability of the dialysis membrane should be tested before and after the release experiment with a solution of the drug to make sure that there is no significant delay in the release. A slow membrane transport may also lead to a violation of sink conditions because of a temporary saturation with drug molecules inside the donor chamber (18). Therefore, the release response for dialysis processes expressed as the membrane permeation rate constant should be determined (3, 21). The significance of the separation time for the sensitivity of the method widely depends on the performance characteristics of the product. Therefore, to avoid the risk of undetected batch-to-batch differences, Figure 1. Illustration of dialysis and reverse dialysis.