Non-liquid matrices (NLMs) encompass a diverse variety of samples from different biological origins, such as tissues, feces, cells, hair, nails, skin, bone, etc. The need to extract analytes of interest (i.e., biomarkers or drugs) from a solid, potentially heterogeneous matrix, distinguishes these samples from homogeneous liquid matrices such as serum, plasma, whole blood, etc. The scientific and regulatory frameworks for the bioanalysis of liquid matrices have been well-established for the past two decades through many white papers, meeting/conference reports, and global health authority guidelines (1,2,3,4,5). Nonetheless, some of the common practices that are mainstays in bioanalysis of liquid matrices cannot be automatically applied to the above-noted NLMs without proper pre-analytical and analytical considerations and implementation of the best practices.

Bioanalysis of NLMs can provide key information, including drug uptake and distribution, target engagement and modulation, and drug exposure at target, that may not be attainable through other approaches (6,7,8). Understanding the drug exposure at the site of action is a major determinant of the therapeutics’ success in phase II clinical trials and their progression to phase III (9, 10). In recent years, bioanalysis of therapeutic molecules and biomarkers in tissue and cell extracts have played an increasingly important role in drug development, particularly for some of the novel modalities targeting nucleic acids, cells, oncolytic viruses, and genes (11, 12). However, bioanalysis of NLMs presents some unique challenges compared to those encountered for liquid matrices. The challenges include but are not limited to the complexity of the structure and properties of the targeted tissues, heterogeneity of analyte distribution in the sample, and potential contamination from blood perfusion (6, 13). To address these challenges, careful consideration is required for both pre-analytical processes (such as sample collection and handling) and bioanalytical processes.

Despite several relatively recent reports pertaining to tissue bioanalysis (6,7,8), there are no clear recommendations on the best bioanalytical practices of NLMs relevant to drug development (8, 14). Moreover, no regulatory guidance has been issued on the bioanalysis of NLMs in support of drug development, though NLM bioanalytical data may be incorporated as supplementary materials in regulatory submissions. Therefore, a consensus on the best bioanalytical practices for NLMs is highly desirable.

This whitepaper is designed to fill this gap by presenting a cross-industry effort to establish an overarching framework for the bioanalysis of therapeutic agents and biomarkers in NLMs. The subsequent sections of this white paper summarize the authors’ recommendations for sample collection, processing, and handling and then propose bioanalytical workflows that can be implemented for both small and large molecules. In addition, the main considerations for bioanalytical method development and characterization and how they are applied to different types of NLMs are captured.

Pre-Analytical Considerations

The development of quantitative bioanalytical methods for NLMs poses unique challenges. For example, NLMs may require specific sample collection, handling, and processing procedures that differ from those for liquid matrices. Additional logistical challenges may arise due to invasive sample collection procedures, the need to preserve analyte stability, and the need to ensure the suitability of the sample for the subsequent intended quantitative analysis. More importantly, the heterogeneity of some NLMs makes it technically difficult to prepare a bulk matrix that is suitable for the preparation of calibration standards, quality controls, and diluent in bioanalytical methods. While several publications describe the processing of tissues and cells, there are no existing guidelines that cover the wide range of NLM types (14). A summary of pre-analytical considerations for NLMs is provided in Fig. 1.

Fig. 1

Pre-analytical considerations

The collection and storage of NLMs depend on the intended assay format, physical state of the sample, and physicochemical properties of the analyte(s) of interest. Studies should be designed to address such pre-analytical considerations to ensure that the NLM collection and processing procedures are compatible with downstream quantitative assay formats. Generation and communication of a detailed study design and corresponding collection and handling procedures helps ensure consistent and interpretable bioanalytical data. A typical workflow for collection and analysis considerations for NLM is depicted in Fig. 2.

Fig. 2Sample Collection and Handling

Sample collection and handling are the first steps in NLM bioanalysis. Sample collection may be invasive and require terminal procedures (in animals), biopsy, or in combination with other surgical procedures. These procedures may occur in clinical or vivarium environments and be performed by personnel who are uninvolved in subsequent bioanalytical activities. Documentation and communication of detailed and specific instructions are therefore critical to avoid issues such as blood contamination and analyte degradation. Typically, tissue or tumor biopsy samples are harvested from specimens with the residual blood removed by blotting and rinsing in an isotonic buffer. Further processing into slices may be done by a refrigerated microtome (15). Cells may also be isolated from samples or whole blood and further processed to enrich specific cell types such as PBMCs (16). Once harvested, samples may be stabilized at the collection site prior to storage, typically via freezing the samples on dry ice, snap-freezing the samples in liquid nitrogen, or adding stabilizing buffers/reagents to the samples (17). Key considerations for NLM sample collection and handling are summarized below:

The experimental design should be clearly documented in study protocols and laboratory procedures that include the sample numbers, site locations, storage capabilities of enrolled clinical sites, and the role and responsibilities of the central lab(s).

Analyte type and any restriction or safety considerations during collection and shipment (e.g., gene & cell therapy, oncolytic viruses) should be clearly described.

Heterogeneity of tumor or tissue samples and consistency in sampling (e.g., multiple biopsies)

Some NLMs, for example, tissue slices, may need to be prepared and stored in multiple aliquots to address the need for multiple analyses, analyte instability, sample loss, or other unforeseen complications during shipment.

The potential impact of contamination from residual blood and neighboring tissues should be assessed.

Specimen shipment logistics and regulatory requirements should also be clearly documented in the study protocol or the corresponding laboratory manual to identify and address potential sources of variability (e.g., domestic vs. international, custom delays for biospecimens potentially containing infectious agents).

Sample Processing

NLMs are often processed to isolate specific tissue regions, enrich cell subtypes (18), or generate cell suspensions. These are then processed to release analytes of interest from the NLM, often by mechanical or ultrasonic processing. Chemical treatment of the NLM sample with lysis buffers/solutions, detergents, or surfactants may also be employed to release analytes from the NLM sample or to maximize analyte recovery. Less aggressive and appropriate methods (e.g., freeze–thaw cycling) may also be employed for specific NLM samples. Homogenization conditions, including buffers, containers, bead types, and mechanical parameters (e.g., total cycles), can be optimized based on the specific analyte or NLM. Temperature control is often used to minimize heat generation during mechanical homogenization, especially for thermally labile or enzymatically released analytes.

The final step in sample processing involves analyte extraction or isolation from NLM homogenates by methods analogous to those developed for liquid biometrics. For small-molecule analytes these include dilution, protein precipitation, liquid–liquid extraction, and solid phase extraction for quantification using LC–MS/MS. For large-molecule analytes these include dilution, immunoprecipitation, and/or enzymatic digestion in preparation for analysis by LBA or LC–MS/MS. The analytes’ physicochemical properties should be considered when selecting an appropriate extraction method. Surrogate matrices or a mixed matrix approach can be adopted when the control tissue matrix is limited or unavailable (19, 20). Examples of sample processing considerations are summarized in Table I.

Table I Sample Processing of Non-liquid MatricesSample Storage and Stability

The assessment of analyte stability associated with sample storage, handling, and processing conditions should occur during method development and prior to the initiation of sample collection (14). NLMs have unique stability challenges, in that spiking into intact tissue or cellular isolates is not easily and quantifiably achievable. It is therefore important to determine how NLM should be processed for storage prior to analysis (Table II).

Table II Sample Storage Considerations in Whole Tissue, Homogenates, and Cell PelletsStability in Tissue

Analyte stability testing in whole tissue (prior to processing) is not feasible. Therefore, tissue samples must be snap-frozen as soon as possible after harvest to minimize any potential analyte degradation. Alternatively, tissues may be homogenized as soon as possible after collection and then frozen for longer-term storage if storage stability in homogenate has been established. Homogenate stability can be assessed by spiking analyte (s) of interest and storing under defined conditions. The assessment of freeze/thaw, short-term, and long-term stability in tissue homogenates can be performed to evaluate stability under different conditions.

Stability in Cell Isolates

The cell isolates can be stored as cell suspensions/pellets or cell lysates. The cell isolates should be stored under frozen conditions as soon as possible after collection to minimize analyte degradation. The stability (short-term, freeze–thaw, long-term, etc.) assessments of the analyte in cell isolates should be performed. If cells are stored in the form of cell lysates. stability can be measured by spiking treatment naïve cell lysates and subjecting the preparation to different stability conditions. If cells are stored in the form of cell suspension/pellets, the stability may be evaluated by collecting and pooling cells from dosed subjects and preparing aliquots of the pooled cells. These aliquots should be analyzed at different time points to assess their stability. For stability to be established, the percent change in concentration compared to its initial concentration (t = 0) must be within pre-defined acceptance criteria.

For NLM specimens, there may be a need to establish temporary storage conditions prior to shipment that are different from the extended storage conditions, based on the clinical site’s capabilities (e.g., -20°C vs. -70°C). Therefore, the details of sample storage and handling should be clearly mentioned in the lab manual and the respective sample stability testing should be assessed during method development/validation to cover the anticipated sample handling conditions. In addition, due to the complexity of the NLM sample, fit-for-purpose method qualification experiments need to be designed based on a feasible stability assessment.

Normalization of NLM Analytical Results for Specific Matrices

For conventional liquid matrices, bioanalytical results are typically reported as either analyte mass per matrix volume (e.g., ng/mL) or molar concentration (e.g., µM). Similarly, to compare NLM samples, the quantitated analyte concentrations need to be normalized based on the sample size. In some cases, the NLM sample can be weighed for normalization. In other cases, the analyte is normalized to another relevant sample parameter (e.g., total protein mass). For cell fractions and isolates (e.g., PBMCs), typically, the number of cells in the sample is determined and results are reported in units of analyte mass or moles per number of cells (e.g., 10^6 cells). For other types of NLMs, a variety of other approaches can be taken for normalization, depending on the specific matrix characteristics and the intended purpose of the analysis.

Table III summarizes approaches for normalization across samples and typical units used for reporting results for various NLM samples.

Table III Non-Liquid Matrices: Approaches for Normalization Across Samples and Typical Units Used for Reporting ResultsVascularity and Perfusion

Tissue vascularity can be variable within the same or different tissue types, and blood contamination can impact the assessment of parameters such as accuracy, recovery, matrix effect, and stability. Accuracy can be impacted due to differences in analyte concentrations between blood and tissue resulting from differences in protein binding and distribution of the analytes in different compartments. In such cases, tissues should be fully perfused and rinsed when possible; if this isn’t possible, the impact of blood components on the relevant analytical assessments should be evaluated.

Tissue Heterogeneity

Tissues are inherently more heterogeneous in their composition than liquid matrices. This heterogeneity leads to differences in analyte distribution and endogenous composition within the tissue, which can complicate the assessment of assay parameters such as recovery, stability, matrix effects, selectivity, and accuracy. Therefore, studies utilizing biopsies or punches should consider analyzing multiple sections of biopsy or obtaining multiple punches. Detailed, and specific identification of the type, size, number, and location of biopsies that are collected for the study should be included in relevant documents (e.g., operations/lab manual). A normal distribution throughout tissue samples can be assumed in many cases unless the preferential distribution is known, and if so, each piece or part of the tissue should be assessed individually. Another approach is collecting and pooling multiple samples, to address concerns about heterogeneity and to ensure a more accurate representation of the sample when assessing certain assay parameters.