Overcoming CMC Challenges In The Development Of Conjugated LNP Platforms For In Vivo CAR-T Therapy

By Sujit Jain, Senior Consultant, Biopharma Manufacturing Sciences and CMC

In vivo CAR-T therapy is rapidly emerging as a transformative approach in cancer treatment, offering a more direct, scalable alternative to today’s complex and costly ex vivo processes.

At the center of this shift are conjugated lipid nanoparticles (CLNPs) — engineered non-viral carriers that deliver mRNA encoding CAR transgenes directly to T cells in the body. By coupling LNPs with targeting ligands such as antibodies, peptides, or small molecules, developers aim to achieve cell-specific delivery with very high precision^1,2,6. However, translating this technology from research to clinical application introduces a new layer of chemistry, manufacturing, and controls (CMC) complexity. Ensuring particle stability post-conjugation, maintaining consistency during scale-up, developing meaningful potency assays, and meeting evolving regulatory expectations all present significant hurdles¹⁰.

This article explores the scientific principles behind CLNP-based in vivo CAR-T therapy, the key CMC challenges impeding its development, and emerging strategies that could enable this promising technology to move from concept to clinical reality^5,8.

A Promising Alternative

CAR-T therapy has transformed cancer immunotherapy, delivering durable responses in hematologic malignancies⁸. Traditional ex vivo CAR-T manufacturing, however, is complex, costly, and slow: patients’ T cells must be harvested, isolated, expanded, and purified in a cGMP facility before being reinfused. This labor- and resource-intensive process limits patients’ access worldwide.

In vivo CAR-T therapy offers a promising alternative by delivering CAR-encoding genetic cargo directly to T cells in the body using non-viral delivery systems^6,9. Unlike ex vivo approaches, this method programs T cells in situ, potentially reducing turnaround time from weeks to days. Early studies suggest this approach could simplify logistics, reduce costs, and broaden accessibility. Various delivery platforms are under investigation, including polymeric nanoparticles, lipid nanoparticles (LNPs), exosomes, and engineered viral vectors such as AAV and lentivirus¹³.

Among these, conjugated LNPs (CLNPs) are gaining traction due to their modular design, manufacturability, and regulatory familiarity, in part stemming from mRNA vaccine development^5,11. By attaching targeting ligands — antibodies, peptides, scaffold proteins, or small molecules — to the LNP surface, CLNPs can selectively deliver CAR transgenes to T cells while minimizing off-target uptake.

Ligands are typically conjugated via PEG-lipid anchors or covalent chemistries to the outer lipid layer, introducing cell-specific binding properties that traditional LNPs lack. This targeted approach has the potential to accelerate clinical translation but also introduces new CMC challenges that must be carefully addressed.

Conjugated LNPs: Novel Formulations

Traditional LNPs are manufactured by encapsulating nucleic acids/genetic payload and delivering them to target region or tissues, but they lack cell specificity (except liver)^1,2,5. CLNPs can offer cell specificity by attaching targeting ligands to the nanoparticle surface. Monoclonal antibodies or fragments, scaffold proteins, small molecules, and peptide or glycan-based ligands are some of these targeting ligands^6,9.

The main goal of formulation development is to deliver a CAR transgene selectively into circulating T cells while minimizing uptake by the liver, spleen, and other non-target tissues. Achieving this consistently and safely is not as straightforward as it may seem even though there is plenty of historic knowledge with these ligands individually^3,4. This requires a tailored CMC approach specific to these particles.

It’s important to distinguish between pre-insertion (ligand added during formulation) and post-insertion (ligand added after LNP formation). The latter often preserves nanoparticle integrity and encapsulation efficiency but introduces its own control and purification challenges.

CMC development for CLNPs rests on three pillars^3,10:

1. Defining critical quality attributes (CQAs)
2. Establishing robust, scalable manufacturing processes
3. Implementing control strategies linking physical properties to biological outcomes

The targeting ligand introduces a new layer of complexity and must be characterized and controlled with the same rigor as the core nanoparticle and nucleic acid payload^4,10.

Addressing Key CMC Challenges In CLNPs

Controlling Ligand Density and Orientation

Consistent and scalable ligand attachment process is still a challenge. Too few ligands may reduce targeting efficiency, while too many can increase aggregation. This may lead to a higher incidence of immunogenicity or rapid clearance or both. Random attachment chemistries would create heterogeneous products⁹, and batch-to-batch consistency could also be a challenge.

Site-specific conjugation strategies — such as engineered cysteines, thiol-maleimide linkages, or click chemistry — offer greater control of ligand density and orientation^6,9. Analytical methods including ELISA (enzyme-linked immunosorbent assay), SPR (surface plasmon resonance), and mass spectrometry are key tools and assays to detect and quantify ligand density and orientation^9,10.

Preserving LNP Integrity Post-conjugation

Conjugation steps can disrupt nanoparticles, impacting the particle core or release of payload^1,2,10. Biodistribution profiles shift with minor changes in nanoparticle integrity. For example, excessive PEG-lipid incorporation can alter surface charge (zeta potential), changing in vivo behavior. Post-insertion strategies, where ligand-PEG-lipids are incorporated after LNP formation, help preserve integrity^9,10. Each batch must be characterized appropriately to ensure payload (mRNA) stability and consistent morphology. Purification steps need to be developed to remove free/unbound ligands. Analytical ultracentrifugation or size-exclusion chromatography can quantify free versus bound species.

Analytical Characterization and Potency Assays

Traditional assays for LNPs, such as particle size and encapsulation, are not enough for CLNPs^1,2,5. Ligand-specific CQAs must be measured, including ligand density, free versus bound ligand, and functional binding affinity. A sequential or layered potency strategy is recommended^8,9.

For example:

• Surrogate assays for ligand density
• Primary T cell transfection assays for CAR expression
• Functional cytotoxic assays to confirm therapeutic activity

Potency assays should also be stability-indicating and linked to product release and comparability testing. Regulators expect demonstration that ligand degradation or loss of orientation correlates with reduced potency^3,4,10,12.

Scale-Up and Reproducibility

Tech transfer of a small-scale optimized process to large cGMP scale is rarely straightforward. Flow rates, mixing dynamics, excipient interactions, and downstream purification conditions can all influence the final particle characteristics¹⁰. For example, differences in shear rate or residence time between microfluidic mixing systems and large-scale impingement or in-line mixers can significantly impact parameters such as particle size distribution, encapsulation efficiency, and RNA integrity. Achieving comparable mixing energy and residence time across scales often requires iterative optimization of flow ratios, buffer composition, and process control strategies.

DoE (design of experiments) approaches help identify critical parameters, while scalable microfluidics/mixing system and process analytical technologies (PAT) provide the consistency needed for robust cGMP manufacturing^3,10.

Stability and Cold Chain

CLNPs are prone to aggregation and degradation, particularly during freeze-thaw cycles^1,10. They are less stable than standard LNPs because ligands can destabilize the particle surface. Stabilizers like cryoprotectants (sugars) or polymeric excipients, as well as lyophilization strategies, are currently being explored^10,11. Long-term stability programs must measure both physical and biochemical properties, and functional potency. Cryo-TEM and differential scanning calorimetry (DSC) can be used to monitor particle morphology and phase transition behavior over time.

Immunogenicity and Off-Target Effects

Despite conjugation, many LNPs are still taken up by the liver and spleen^1,2,9. The unintended expression of CARs in non-T cells represents a major safety risk. Targeting moieties/ligands themselves can also trigger immune responses. De-immunized ligands and smaller scaffolds may mitigate these risks, as well as transient mRNA-based expression rather than permanent integration^6,8,9. A variety of in silico and in vitro testing programs are available to evaluate the immunogenicity potential and engineer a targeting moiety sequence to reduce the potential for immunogenicity and improved stability.

Regulatory Expectations and Comparability

CLNPs bring gene therapy, nanomedicine, and biologics together. Regulators expect detailed control over conjugation chemistry, ligand characterization, and comparability across process changes and tech transfers. Guidance is evolving under the FDA’s CMC for Gene Therapy INDs and EMA’s Horizon Report on Nanotechnology based medicinal products for human use¹⁴.

Early agency engagement and CMC strategies like separate leaflets for backbone LNP, payload, and ligand could help spur faster review cycles and approval^3,12. This modular documentation allows developers to modify ligands or payloads without reopening the entire dossier.

Emerging Solutions

Several strategies are emerging to overcome these challenges. Platform approaches standardize the backbone lipid nanoparticle, so ligands as well as payloads can be swapped without significant process development/optimization^2,6,9. Next-generation analytics, such as single-particle analysis and high-throughput flow cytometry, provide deeper insights into heterogeneity^9,10.

Manufacturing innovations, including continuous LNP processing, SPTFF (single pass tangential flow filtration), and in-line PAT tools, support real-time control of CQAs such as particle size and ligand density¹⁰. These technologies directly address scale-up and comparability bottlenecks while improving reproducibility.

Ultimately, success will depend on cross-functional collaboration between CMC, bioanalytical/non-clinical, and regulatory teams from the earliest stages of development.

Conclusion

Conjugated LNPs represent one of the most promising delivery systems for in vivo CAR-T therapy. They offer the potential to simplify manufacturing, broaden access, and reduce costs, but their development is technically demanding. From ligand conjugation and stability to analytical complexity and regulatory expectations, each challenge requires scientific and disciplined innovation. If these hurdles can be overcome, conjugated LNPs may transform in vivo CAR-T therapy and open the door to more accessible, scalable cancer immunotherapies.

References:

1. Hou X. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021.
2. Kulkarni JA. et al. Lipid nanoparticle technology for nucleic acid therapeutics. Nat Nanotechnol. 2023.
3. FDA Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy INDs. 2020.
4. European Medicines Agency: Reflection Paper on Nanomedicines. 2021.
5. Verbeke R. et al. The dawn of mRNA vaccines: The lipid nanoparticle breakthrough. Nat Drug Discov. 2021.
6. Miller JB. et al. Targeted lipid nanoparticles for in vivo gene editing. Nat Biotechnol. 2020.
7. Buschmann MD. et al. Nanomaterial delivery systems for mRNA vaccines. Nat Rev Drug Discov. 2021.
8. Patel S. et al. Challenges in developing in vivo CAR-T therapies. Mol Ther. 2022.
9. Zhang X. et al. Engineering targeted lipid nanoparticles for cell-specific delivery. Adv Drug Deliv Rev. 2022.
10. Sabnis S. et al. Manufacturing considerations for LNP-based therapeutics. Mol Ther. 2018.
11. Pardi N. et al. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov. 2018.
12. EMA/FDA joint workshops on nanomedicines and gene therapies, 2022–2023.
13. Deng W. et al. Advancements and challenges in developing in vivo CAR T cell therapies for cancer treatment, EBiomedicine, 2024 Aug:106:105266
14. EMA EU-IN Horizon Scanning Report - Nanotechnology-based medicinal products for human use

About The Expert:

Sujit Jain is a pharmaceutical and life sciences professional with extensive experience in process development, external manufacturing, and CMC operations across biologics and advanced therapy programs. His work has involved managing technology transfers, CDMO partnerships, and clinical supply chains, with a focus on bridging development and manufacturing to enable reliable, scalable delivery of complex therapies.