Overview of Bioanalytical Methods for Cell Therapy Products

Overview of Cell and Gene Therapy Products

Cell and gene therapy (CGT) can be broadly categorized, based on product format, into ex vivo gene therapy and in vivo gene therapy. In general, ex vivo gene therapy primarily involves cell-based gene therapy, which entails isolating the patient’s target cells, genetically modifying them outside the body using techniques such as gene regulation, replacement, addition, or deletion to alter their biological properties, and then infusing these modified cells back into the patient as a live-cell therapeutic approach for treating human diseases. In contrast, in vivo gene therapy mainly refers to traditional—or narrowly defined—gene therapy, in which therapeutic agents are directly administered into the patient’s body to intervene at the genetic level, modulating or correcting gene expression. Ex vivo (cellular) gene therapy products in clinical practice primarily include immune-cell gene therapies, stem-cell gene therapies, and other cellular gene therapies; whereas in vivo gene therapy mainly comprises viral-vector–based gene therapy, lipid-vector–based gene therapy, and nucleic-acid–based gene therapy. However, in vivo gene therapy products are beyond the scope of this post; readers are encouraged to refer to other related posts on the Kanwhish official WeChat account for more information.

Immune-cell gene therapy involves harvesting a patient’s autologous or allogeneic immune cells, culturing them ex vivo, and genetically modifying and engineering them to acquire targeted cytotoxic activity. The modified cells are then infused back into the patient, where they exert therapeutic effects by directly targeting and killing malignant or pathogenic cells, modulating the immune system, attenuating immune tolerance, and enhancing immune responses. Based on the specific immune cell types used and the nature of the genetic engineering employed, the main categories of immune-cell gene therapy include CAR-T, CAR-NK, and T-cell receptor–engineered T cells (TCR-T). However, unmodified cell therapies such as tumor-infiltrating lymphocytes (TILs) and cytokine-induced killer cells (CIKs) are not classified as immune-cell gene therapy. The most clinically mature form of immune-cell gene therapy is anti-CD19 and anti-BCMA CAR-T cell therapy for lymphoma; several CAR-T products have now been approved for marketing by regulatory authorities including China’s NMPA, the U.S. FDA, and the European Medicines Agency (EMA). Another example is Zalmoxis®, an allogeneic T-lymphocyte suicide-gene–based immunotherapy that was previously used as an adjunct treatment for graft-versus-host disease following hematopoietic stem-cell transplantation but has since been withdrawn from the market.

Stem cells possess the properties of self-renewal, multipotent differentiation, and excellent tissue compatibility, making them suitable as cellular carriers for gene therapy agents and endowing them with broad application prospects in regenerative medicine and cell and gene therapy (CGT). Gene therapy products based on human-derived stem cells and their derivatives involve autologous or allogeneic human stem cells that undergo a series of ex vivo manipulations—including isolation, separation, purification, culture, expansion, gene editing/modification, directed differentiation, cryopreservation and thawing, and transportation—to generate clinical trial–ready formulations. These formulations entail editing and correcting defective genes in the stem cells before reinfusion (or implantation) into the patient, thereby enabling intervention and treatment of genetic diseases. Stem cell–based gene therapy products not only exhibit the characteristics of cell therapies but also those of gene therapies, which imposes higher requirements on clinical trial design. However, current clinical data are predominantly derived from single-center studies with small sample sizes and focus on rare diseases, presenting numerous challenges for the development of such therapeutic products as well as for assessing their safety and efficacy. Consequently, rigorous and comprehensive bioanalytical testing is essential to ensure the smooth conduct of clinical trials involving stem cell–based gene therapy products and constitutes an indispensable component of the development process. Stem cell–based gene therapy is currently concentrated on rare diseases and neurodegenerative disorders; among the stem cell–based gene therapy products approved for marketing in Europe and the United States are Strimvelis® for the treatment of adenosine deaminase–severe combined immunodeficiency, Zynteglo® for transfusion-dependent β-thalassemia, Skysona® for early-onset cerebral adrenoleukodystrophy, and Libmeldy® for early-onset metachromatic leukodystrophy.

Bioanalytical Testing of Cell Therapy Products

After CGT products are administered, cells may differentiate into both intended and unintended cell types and undergo interconversion among these types; in particular, therapeutic products composed of heterogeneous cell subpopulations may also acquire additional mechanisms of action (MOAs). On the other hand, since cell-based therapies are delivered systemically via the circulatory system, cells can distribute throughout various tissues, and even when transplanted to a target tissue site—such as the bone marrow—unintended migration may occur. Therefore, it is essential to conduct research on the biodistribution, homing, and tracking of cell-based therapies. Moreover, when CGT products integrate into the genome, uncontrolled gene editing (i.e., genomic mutations) may occur, leading to the activation or inactivation of critical genes and potentially triggering carcinogenesis. CGT products thus exhibit characteristics of both cell therapies and gene therapies, with the associated potential risks. Consequently, bioanalytical characterization in clinical trials constitutes a key component of CGT product development, as comprehensive clinical bioanalysis enables:

Elucidate the pharmacokinetic (cellular) kinetic characteristics of CGT products in the human body to provide supporting evidence for the selection of routes of administration and dosing regimens.

Confirm the MOA of CGT products, the purpose and function of genetic modification, and the expression levels of transgenic proteins, and clearly define the therapeutic efficacy and mechanism of action of CGT products.

Based on potential biological risk factors, predict the spectrum of immune response characteristics following human administration, identify clinical monitoring indicators for adverse immune reactions (both adaptive and innate immunity), and provide a reference basis for developing clinical risk-control measures.

Therefore, it is recommended to conduct comprehensive bioanalytical studies to collect the data and information necessary for evaluation, thereby establishing that the developed CGT product exhibits a reasonable and acceptable benefit–risk profile from a bioanalytical perspective and providing evidence-based support for clinical trial design and risk management strategies.

Pharmacokinetic (Cellular) Kinetics Analysis

The pharmacokinetic (cellular kinetic) characteristics of CGT products differ markedly from those of conventional small-molecule drugs and large-molecule biologics, and most CGT products are difficult to evaluate using traditional absorption–distribution–metabolism–excretion (ADME) classification frameworks. Pharmacokinetic studies of CGT products should elucidate the in vivo biological processes and associated biological behaviors of cell-based therapies, and, based on the type and specific features of the cell therapy product—particularly immune-cell gene therapy and stem-cell gene therapy—as well as the research objectives and the clinical relevance of the analytical endpoints, establish appropriate bioanalytical methods and perform the requisite method validation. Such studies should primarily focus on parameters such as changes in the number of target cells following administration; monitoring of cell viability and phenotypic differentiation (e.g., cell phenotype and functional biomarkers); duration of action (including pharmacokinetic constants such as area under the plasma-concentration–time curve, peak concentration, and time to peak); functional activity during the expected survival period (or surrogate markers thereof); in vivo biodistribution; ectopic localization; tissue tropism and migration; expression and/or secretion of biomolecules; and interactions with host tissues. In some cases, these interactions may also encompass tissue responses elicited by non-cellular components of the cell therapy product and by secreted bioactive molecules.

Pharmacokinetic assays for CGT products typically recommend employing multiple analytical approaches during method development to evaluate and monitor the in vivo behavior of target cells. Given that immune-cell gene therapy and stem-cell gene therapy products are engineered through genetic engineering techniques, both quantitative real-time PCR (qPCR) and flow cytometry can be used concurrently for analysis. These two techniques respectively quantify the copy number of the exogenous transgene and the changes in the number of cells expressing the desired phenotype (e.g., CAR+ or transgene-positive), thereby enabling a more comprehensive assessment of the expansion and survival of cell-therapy products in vivo. When designing and validating pharmacokinetic assay methods, qPCR results can be interpreted as representing the “total” product burden in the body, whereas flow-cytometry results reflect the “effective” cell population (Figure 2). Widely used PCR-based methods include qPCR and digital PCR (dPCR), which are employed to monitor the copy number of the transgene in vivo. The process begins with the design of highly specific primers and probes targeting the sequence of the transduced exogenous gene—typically spanning regions such as the scFv or the junction between two gene segments—followed by the preparation of genomic DNA standards, the selection of appropriate reference genes (e.g., ACTIN or CDKN1A), and the application of efficiency correction factors based on qPCR reaction kinetics and gDNA input. Ultimately, this enables the determination of the CAR transgene copy number per unit of gDNA in the subject’s blood samples. To assess the analytical sensitivity and reproducibility of qPCR for CAR-T cells, digital PCR can be used for absolute quantification of CAR copies, with the resulting values serving as a reference standard for qPCR assays. Digital PCR also enhances the detection of rare events and reduces assay variability; when sampling is conducted over sufficiently long time periods, the abundance of the target analyte may become very low, necessitating highly sensitive analytical methods, in which case a digital-PCR–based approach should be considered. For flow-cytometry–based methods, the primary focus during development and validation is the generation of suitable positive-control cell samples; ideally, the positive control would be the cell-therapy product itself, but this is often impractical. Therefore, the goal in constructing positive-cell standards is to achieve the closest possible match to the intended use sample. In addition, the selection and preparation of critical reagents and the optimization of method sensitivity are key priorities during the early stages of product development. For example, CAR-T cell products can be characterized pharmacokinetically by quantifying CAR+ cells; although flow cytometry may sometimes exhibit slightly lower analytical sensitivity than PCR, its output is expressed at the cellular level, which aligns more closely with the biological concept of “cellular dynamics” and allows simultaneous analysis of dynamic changes across different cell subpopulations. Furthermore, flow-cytometry–based method development does not require sponsors to provide core CAR sequence information, which represents a significant advantage.