4.1 Bioanalytical Considerations for Various Biological Matrices
Relatively routine methods are established for sample processing and testing in well-characterized matrices such as plasma, serum, and urine. For biological drugs, the conventional delivery routes are subcutaneous, intramuscular, and intravenous. However, pursuit of novel administration routes and novel bioconjugates entails the integration and analysis of different biological matrices, which presents unique bioanalytical challenges.
Bioanalysis in biological matrices other than plasma, serum, or urine can offer important information on the distribution of biological drugs, which can inform mechanism of action, ADME, and/or safety considerations. For biotherapeutics with a novel route of administration, bioanalysis may be needed for the tissue(s) from corresponding absorption site(s). For ADCs, tumor distribution of ADC and released warhead can establish the exposure–efficacy relationship and may contribute to pharmacokinetic/pharmacodynamic modeling. Tissue bioanalysis is conducted mostly in preclinical studies, especially during the lead selection and lead optimization. Depending on the specific project needs, assays supporting tissue bioanalysis may vary. For most cases, a quantification assay for the drug candidate is needed. Occasionally, additional assays of major metabolites may be of more interest.
For biological drugs, the main challenge with tissue bioanalysis comes from the analyte itself. In small-molecule bioanalysis, the tissue can be disrupted in a thorough manner followed by direct precipitation or further extraction procedures using organic solvents. However, biotherapeutics typically cannot withstand such rough sample preparation procedures and maintain the capability to selectively bind to the capture reagent, which is frequently required for bioanalysis of biologic drugs. Special buffers known to retain the structural integrity of the biological drug, such as radioimmunoprecipitation assay buffer or tissue protein extraction reagent, are often used in the extraction of the analyte [
124,
125]. These buffers, although gentle enough to preserve the structural integrity of the macromolecule, may result in incomplete tissue disruption and affect the extraction recovery. Therefore, in addition to tissue weight, normalization against total protein concentration may also be considered when developing methods for extracting biological analytes from tissues. On the other hand, the small-molecule format of tissue preparation can still be utilized if the analyte of interest is a small molecule, such as free warhead for bioconjugates, or a structurally modified peptide. In some cases, capture of the biotherapeutic analyte is not always necessary, as has been shown for the direct digestion approach for a cocktail of co-dosed antibodies administered at very high doses for the prevention of coronavirus disease 2019 (COVID-19) [
126] and post-pellet digestion followed by solid-phase extraction (SPE) clean-up [
127] of mAbs. Both of these methods have been applied to serum samples. It would be interesting to consider the application of such approaches to the bioanalysis of therapeutics from tissues that can benefit from harsher extraction conditions. Such approaches would require very careful evaluation of highly selective surrogate analyte peptides and/or extensive sample clean-up procedures.
In addition to tissue bioanalysis, NLF has been gaining more attention during the COVID-19 pandemic. Nasosorption™ FX·i is an example of a device that absorbs the biofluid from the nasal mucosa [
128]. During bioanalysis, an elution solution and a device strip are added to a tube. Analytes are extracted by vortexing and centrifugation of the device stripe. This extraction process must be well characterized to establish adequate and consistent recovery [
129].
Although bioconjugate analysis in unique matrices presents a clear challenge, past success in both small- and large-molecule sample preparation for pharmacokinetic/pharmacodynamic analyses can help guide bioconjugate analytical efforts. An appropriate sample preparation technique is essential to ensure the analytical performance of the method. Saliva and sputum are heterogeneous viscous matrices. This challenge has been addressed with the use of reducing agents such as dithiothreitol, which reduce protein disulfide bonds, making the matrix more homogeneous and reducing viscosity, enabling standard liquid-handling procedures [
130]. Tissues such as lung or skin present difficulties because of the elastic connective tissue. Physical disruption methods, such as rotor stator homogenizers or grinding with a mortar and pestle in liquid nitrogen, are established methods that can potentially benefit bioconjugate analysis, but they are inherently limited because of throughput. Cryogenic ball mills offer a viable solution with higher throughput [
131,
132]. Ultimately, it may be necessary to refine such procedures based on actual method performance.
In certain cases, bioanalysis of analytes present in biological matrices at very low quantities is required. Some examples are NLF, sputum, bronchoalveolar lavage, bone marrow aspirate, tears, and cerebrospinal fluid. When working with matrices that are difficult to obtain, a surrogate matrix approach may be warranted. Common matrices for analysis are typically readily available from commercial suppliers. However, some of the already mentioned rare matrices can be difficult or expensive to obtain. In such cases, a surrogate matrix should be employed [
133]. Wakamatsu et al. [
134] proposed a strategy for surrogate matrix selection for ligand-binding and LC-MS assays. For a surrogate matrix to be deemed appropriate to support quantification of a given analyte, acceptable precision, accuracy, and parallelism must be demonstrated. Matrix effect and extraction recovery evaluations in original and surrogate matrices are also required for validation. For exploratory studies where the original matrix is unavailable, full validation may be unnecessary and/or infeasible [
135].
4.2 Absorption, Distribution, Metabolism, and Excretion
In contrast to small molecules, bioconjugates are structurally complex. This complexity increases for ADME studies and demands more bioanalytical methodologies necessary to support them. Mechanisms of small-molecule drug metabolism have been well established through decades of research, and the utility of this knowledge is not lost for bioconjugates, particularly for ADCs that contain a therapeutic warhead, which, as a free entity, adheres to small-molecule clearance mechanisms with similar toxicology potential [
136]. However, an intact bioconjugate behaves more like a large molecule and adheres to proteolytic degradation pathways recycling the peptide structure into amino acids [
137,
138]. The end result is an assortment of metabolic products ranging from small-molecule warhead metabolites to an intact bioconjugate requiring analytical support to establish therapeutic stability and a toxicology profile [
139,
140]. Distribution creates analytical challenges because of the targeted nature of many bioconjugate structures. Bioconjugate structures are usually highly targeted as the antibody structure enables the therapeutic to bind to specific proteins [
139]. As a consequence, bioconjugate distribution will be much higher in target than in off-target tissues. Bioanalytical strategies designed to assess tissue distribution must be able to function across multiple matrices and cover a greater range of concentrations to quantify bioconjugates across target and off-target tissues. Positron emission tomography approaches can be highly complementary to traditional bioanalytical approaches in assessing the biodistribution of bioconjugates, enabling richer temporal sampling of their distribution because of the inherently noninvasive imaging approach [
141].
Bioconjugates also create unique absorption considerations. The majority of bioconjugate therapeutics on the market and in development are injected to overcome absorption challenges associated with oral or inhaled delivery routes and to mitigate expected toxicities in case of ADCs. However, as discussed, excipients themselves should be evaluated for toxicity liability, and the excipient itself may interact chemically with the bioconjugate, creating further structural complexity that bioanalytical methodology must encompass for effective quantification.