Analytical Considerations for Gene-Modified Hematopoietic Stem and Progenitor Cell Therapies: Part 2 — Starting Materials and Drug Substances

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This two-part review provides high-level analytical development considerations for ex vivo, genome-modified hematopoietic stem and progenitor cell (GM-HSPC) products derived from primary donor cells. Part 1 in BPI’s May 2024 issue addresses analytical controls for in-process drug substances and drug products. Here in Part 2, we take a step back to examine concerns for HSPC source materials. Look to other recently published reviews for a broader discussion of chemistry, manufacturing, and controls (CMC) for GM-HSPCs (19, 20) and for development considerations with gene-edited pluripotent stem cells (PSCs) (21). Note that we use the term genome modified in a generic sense herein to include products that are manufactured by means of viral-vector transduction (typically by lentiviral vectors (LVVs)) and those subject to genome editing by such means as a system based on clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9).

Analytical Controls for Starting Materials and Drug Substances

To ensure GM-HSPC quality, thorough analytical control strategies should be implemented that include a phase-appropriate set of in-process, characterization, and release tests to monitor both manufacturing processes and drug products. Figure 2 depicts a generic GM-HSPC manufacturing process, beginning with the introduction of cellular starting material, which is controlled through establishment of donor eligibility criteria and starting-material testing. Genome-modification reagents — e.g., nucleases, single-guide RNA (sgRNA), and viral vectors — usually are classified as drug substances and thus are subject to release testing before their entry into the manufacturing process. Control of both cellular starting material and genome-modification reagents are discussed below. Note that GM-HSPC manufacturing processes often proceed uninterrupted, and often there is minimal or no testing of cellular drug substances.


Many starting materials and critical reagents for products such as GM-HSPCs can be of varying quality and/or the product of bespoke manufacturing, themselves. Thus, it is necessary to place significant emphasis on the analysis of cellular starting materials and genome-modification reagents. For these complex products, investing up front in a comprehensive analytical approach might help to accelerate development and mitigate later-stage risks.

Cellular Starting Materials: Most GM-HSPC processes — including those used to make the six commercially approved products to date (see Part 1, Table 1) — rely on the acquisition of autologous cellular starting material (66–71). Allogeneic products have entered clinical development recently (72). Such efforts include ongoing work to establish “universal” HSPCs derived from PSCs. Those products are beyond the scope of this review; considerations for their development were described by Morse and Mack in 2023 (21).

Whether derived from an autologous or allogeneic source, control of cellular starting material follows two parallel pathways: donor eligibility determination and cellular testing (Table 3). Questionnaires, medical examinations, and viral testing serve to establish donor eligibility (73, 74). Donor testing typically includes testing for hepatitis, human immunodeficiency virus (HIV), and other pathogens as well as prion contagions. After those screenings, eligible donors are subjected to a mobilization regimen that enables CD34+ stem cells to move from the donors’ bone marrow to their peripheral blood (75, 76). Thus “mobilized,” they provide peripheral-blood donation that is subjected to leukapheresis, extraction of white blood cells from the peripheral blood (9). Alternatively, CD34+ stem cells may be acquired by extraction of bone marrow directly from the hip bone (9).


Cellular testing begins upon receipt of leukopaks or other starting material at the biomanufacturing site. Such testing typically focuses on the quantity and health of target CD34+ cells through testing of viability and measurement of both total nucleated cell count and the percentage of CD34+ cells present. When possible, limits for such tests should be based on manufacturing capability and potential effects on critical quality attributes (CQAs). Limits are likely to be established based on a minimum viability to provide a product of sufficient quality and a minimal cell count and CD34+ percentage to ensure adequate dosing. Additional tests — e.g., safety testing or analysis of specific cell phenotypes — also might be considered necessary for a GM-HSPC program.

Precedents have been established for several approaches to genetic modification of HSPCs (77). For our purposes, we consider genome-modification reagents — including viral vectors, nucleases, and sgRNA — to be active pharmaceutical ingredients (APIs) or drug substances and therefore subject to an appropriate level of analytical control (78). Release of each reagent should be contingent upon demonstration of sufficient purity, safety, and potency.

Nucleases: Release specifications for nucleases such as the Cas9 protein should be set to ensure both the consistency of the manufactured nuclease and the safety, purity, and potency of the corresponding GM-HSPC drug product. Table 4 lists typical assays for nuclease release. The list is not exhaustive and applies only to the given nuclease (protein) itself, although similar principles apply to mRNA as well.


Perhaps the most important nuclease attributes are safety and activity, which are key to ensuring quality of GM-HSPC drug products. Safety test panels include compendial sterility, mycoplasma, and endotoxin assays (or equivalents); activity assays might be designed to measure the ability of a nuclease to cut (or otherwise modify) template DNA. Evaluation of purity typically requires a method such as high-performance liquid chromatography (HPLC) to measure the percentage of intact, full-length nuclease molecules. Impurity determination often relies on multiple assays to evaluate host-cell proteins (HCPs), host-cell DNA, and nuclease degradants/aggregates present in a product sample.

An increasing number of good manufacturing practice (GMP)–quality nucleases have become available commercially, providing a useful route to minimizing cost and complexity relative to internal manufacturing (79–81). However, to ensure that suppliers can support GM-HSPC programs throughout development, product sponsors should exercise appropriate oversight (e.g., vendor management programs, audits, and so on) before integrating “off-the-shelf” options. For early stage clinical development, that includes robust platform assays and specifications that are appropriate for manufacturing HSPC drug products. For later-stage development — including studies enabling licensure of clinical material and commercial manufacturing — sponsors should ensure that their suppliers have strong analytical validation programs in place. In such later phases, sponsors also need their own appropriate quality systems and risk-assessment procedures with associated documentation of all changes in production processes and analytical methods.

Single-Guide RNA: Given the critical nature of sgRNA sequences in determining CRISPR/Cas9 specificity, their purity and identity are considered to be critically important to PQAs. Ion-pair reversed-phase HPLC (IP-RP HPLC), which separates oligonucleotides based on their length and charge, is a standard method used for measuring sgRNA purity (81). Note, however, that both molecular length and commonly used chemical modifications such as phosphorothioate linkages can present significant challenges in the use of chromatographic approaches (82). Mass-spectrometry (MS) and next-generation sequencing (NGS) approaches also can be used for establishing sequence purity.

Recent publications demonstrate that LC-MS approaches can be used to demonstrate sequence identity, to detect sequence modifications, and possibly to establish the sequence purity of targeted regions of sgRNA molecules (83–85). However, LC-MS has yet to sequence full-length sgRNA quantitatively. NGS analysis theoretically should apply to quantitative sequencing of sgRNA but for evaluation of chemical modifications, although biases during amplification can complicate the technique’s reliability in quantitating sequences (86). Such effects should be evaluated before implementation of NGS assays for sgRNA purity assessment. Table 5 lists assays usually found on sgRNA release-testing panels.


Ribonucleoprotein (RNP): Neither sgRNA nor RNA-directed nucleases have significant biological activity in isolation. Rather, those components combine to form RNP complexes that modify DNA. Thus, regulators expect sponsors to provide at least some characterization of those complexes. For cases in which RNPs are prepared ex vivo, sponsors at minimum should implement both purity and activity assays to characterize those complexes. Purity assessments may include analysis of both the proportion of intact RNP and proportions of sgRNA and nuclease present (87); activity assays probably can be established in the same manner as noted above for the release of sgRNA or nuclease.

Viral Vectors: Lentiviral vector (LVV) manufacturing poses several challenges, including needs for consistent production (e.g., by stable and high-yielding producer cell lines) and for highly accurate titer assays for quantifying LVV concentrations and safety (88–89). Unlike other viral vectors, LVV production poses difficulty to those companies attempting to use stable cell lines (90). Thus, most conventional practices use transient transfection of adherent cell lines, which has presented difficulties in scale-up (91). That said, establishing stable cell lines would eliminate steps required for transient transfection and enable continuous and consistent vector production (92).

Analytical control of LVVs involves a comprehensive and well-established set of methods and techniques for ensuring the quality, purity, and functionality of vector preparations. Several methods are used in quantifying viral titers to ensure the appropriate dosage for intended applications. Those methods can be categorized broadly into functional and nonfunctional approaches (93). Nonfunctional (physical) titer methods include assessments of p24 capsid protein and lentiviral RNA levels. A significant disadvantage of such methods is the potential for overestimating vector titers through quantification of protein or RNA coming from both functional and defective vector particles (93). Infectious titer assays use real-time quantitative polymerase chain reaction (RT-qPCR) to measure mRNA expression from transduced cells. Such a functional approach is considered to be more accurate for determination of functional titers (94).

Additional key aspects of analytical control include evaluating the absence of contaminants such as HCPs, nucleic acids, and other foreign particles; verification that LVVs have the correct genetic material and maintain their intended identity; assessment of the vectors’ ability to achieve desired transduction efficiency; and analysis of LVV integration patterns within a host genome to evaluate the risk of insertional mutagenesis (95). Table 6 is a typical release-testing panel for LVVs used in manufacturing GM-HSPCs.


With measures such as splitting of the LVV genome into separate plasmids and partial deletion of the 3' long-terminal repeat (LTR) reducing risks of replication competence, the safety of LVVs has improved over the years, thus minimizing the associated patient safety risks (96). The recommended assay for assessing replication-competency of viruses involves coculture with indicator cells and subsequent evaluation of the presence of viral protein and/or DNA sequences. Nevertheless, alternative rapid methods can be used for detecting replication-competent LVV if their equivalence or superiority to the traditional coculture assay can be demonstrated (97).

Rising to the Challenges

Whether manufactured through viral-vector–mediated gene delivery, nuclease-mediated editing, or both, genome modification of HSPCs represents a significant advancement in the potential to cure diseases that otherwise have suboptimal or no currently available treatment options. Building on a substantial therapeutic legacy of HSC transplants, these new therapies are complex to manufacture and require broad and deep analytical support to ensure adequate and consistent product quality (13).

GM-HSPCs face two key analytical challenges: First, the components used to manufacture them are often bespoke and require significant analytical oversight. Second, as detailed in Part 1 of this review, the broad range of materials used to manufacture a GM-HSPC can include proteins, nucleic acids, viral vectors, and cellular materials. Each of those requires the development of its own bespoke analytical approach, including specific analytical tools and methods. Challenges associated with appropriate characterization of GM-HSPC products will increase as the field matures, with the potential addition of new gene-editing techniques — such as base and Prime editing and gene writing (62–64) — as well as in vivo targeting approaches (65).

Thus, GM-HSPC sponsors are advised to put significant thought into the development of appropriate analytical control strategies for each of their candidate therapies. Doing so can help maximize the probability of regulatory, technical, clinical, and commercial success, thus helping to maximize the likelihood of each candidate achieving its therapeutic potential.


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Corresponding author Brent Morse is a principal consultant, and Alicja Fiedorowicz is an analytical consultant in cell and gene therapy, both at Dark Horse Consulting Group near Boston, MA; [email protected].

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