Points To Consider For Assessing Lipid Quality For Lipid Nanoparticle Manufacturing

By Mo Heidaran, Cellx Inc.

In the last few decades, we have made significant progress in our understanding of how lipid nanoparticles (LNPs) can be used to deliver different therapeutic cargos ex vivo or in vivo. Today LNPs are used to deliver a variety of different agents, including anti sense oligonucleotides (ASO), guide RNA (gRNA), mRNA, and short-interference RNA (siRNA) to modulate, restore, or regulate function of a target gene ex vivo or in vivo.

Table 1 shows diverse applications of LNPs for ex vivo editing of cellular product and in vivo editing applications and delivery of other therapies, including siRNA, and ASO in two broad categories of prophylactic and therapeutic areas. Despite a wide array of technologies using LNPs, however, currently there are only a handful of RNA/LNP-based drugs approved by the FDA. These include amyloidosis siRNA and ASO gene therapies Onpattro, Amvuttra, and Tegsedi, and Moderna’s Spikevax and Pfizer’s Comirnaty COVID-19 mRNA vaccines.

Table 1: Summary of LNP applications

Products including lipid to formulate LNPs	Prophylactic vaccine	Therapeutic cancer vaccines	Gene modulation	Gene correction
LNP/Cas9mRNA/gRNA (ex vivo and in vivo)				X
LNP/mRNA (in vivo)	X	X
LNP/siRNA (in vivo)			X
LNP/oligonucleotides (ASO) (in vivo)			X

 

But First, The Difference Between Prophylactic And Therapeutic LNPs

Before getting into the reason for this article, it is important to distinguish therapeutic versus prophylactic uses of LNPs for mRNA delivery.

The differences start in the offices within CBER in which each product category is regulated. Prophylactic vaccines that are intended to prevent infectious diseases, such as vaccines, are regulated by the Office of Vaccine Research Review (OVRR). In contrast, most of therapeutic cancer vaccines that are intended to have anti-tumor properties and other products identified in Table II, except for siRNA and ASO products using LNPs, are regulated in the newly organized Office of Therapeutic Product (OTP).

The second area of major difference is the target population. It is important to point out that prophylactic vaccines are commonly administered to healthy individuals while therapeutic vaccines are almost invariably administered to patients suffering from life-threatening conditions. In this context, one should consider the safety issues related to the historical use of LNPs in terms of dosage, frequency of administration, and health of the target population.

The third area of distinction is related to mechanism of action (MOA) of prophylactic products versus therapeutics. For prophylactic products, stimulation of innate and adaptive cell immunity could potentially enhance the efficacy of prophylactic vaccines like SARs-CoV2. However, the immune response elicited by LNP and or nucleic acid complexes may not be necessarily desirable for therapeutic applications, such as, for instance, when used to correct a genetic disease ex vivo or in vivo, particularly when such therapy is used to treat diseased populations potentially having multiple confounding comorbidities.

The fourth area that pertains to the total dosage of LNP used is related to the route of administration and frequency of administration. For prophylactic vaccine the preferred route of administration is intramuscular (IM), while  in vivo gene therapy genome editing applications are likely to be intravenous (IV), requiring a larger dosage of LNP delivered (see Table 2 for a summary of differences).

Table 2: LNP Examples

Product and jurisdictional center to regulate at FDA	Examples	MOA/Target cells	Route of administration	Frequency of administration	Target population	Lipid designation based on BLA review memos/dossier
LNP/mRNA (Prophylactic)1, 2 CBER/OVRR	SARs-CoV2	Immune response (prevention of Infection) immune cells	IM	30-100 ug3	Healthy individuals	Novel Excipient1 or Starting Materials2
LNP/mRNA (Therapeutics) Cancer Vaccines4 CBER/OTP	Neoantigen cancer vaccines	Immune response (anti-tumor response) immune cells	IV	Repeated administration	Cancer patients	NA
LNP/Cas9/gRNA (Therapeutics)5 CBER/OTP	In vivo genome editing Verve-101	Gene editing (correction of gene in vivo) hepatocytes	IV	One time administration 0.3, 0.45, 0.6 mg/kg.	Patients with genetic diseases	NA
LNP+/Cas9/gRNA (+ionizable lipid plus GalNAc)5 CBER/OTP	Verve 102	Gene editing (correction of gene in vivo) hepatocytes	IV	Not available	Patients with genetic diseases	NA
LNP/siRNA6, 7, 8 CDER	In vivo gene silencing Alnylam Pharmaceuticals vutrisiran (Amvuttra)	Gene silencing hepatocytes	SC	25 mg dose every 3 months	Patients with hereditary variant transthyretin amyloidosis	Novel excipient
LNP/siRNA Dlin-MC3-DMA8, 9 CDER	Patisiran (Onpattro)	Gene silencing hepatocytes	IV	0.3 mg/kg once every 3 weeks for under 100 kg or 30 mg/kg every 3 weeks for over 100 kg	Patients with hereditary variant transthyretin amyloidosis	Novel excipient

Examples of prophylactic vaccines versus therapeutic cancer vaccines as compared to LNPs for delivering genome editing agents. This table is provided for illustrative purposes to highlight differences between different products being investigated or approved using LNPs. IM: Intra muscular, SC: Subcutaneous, IV: Intravenous

Characterizing LNPs And Their Purity Attributes

LNPs consist of four lipid types, including ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids.

The ionizable lipid enables the encapsulation of RNA and facilitates the transport of RNA to the cytoplasm through its tertiary amine groups. Phospholipids and cholesterol aid the LNP complexes to achieve an endosomal escape, which is shown to be important for their function.^{10, 11}

On the other hand, enhancing the half-life of LNPs prolongs their circulation time in the body. There are several well-known adverse events associated with LNP-containing products. Some of these adverse effects include anaphylaxis and complement activation-related pseudoallergy reaction (CARPA), including IgE-mediated allergy, non-IgE-mediated allergy, and autoimmune disease. It is still unclear whether there is a causative connection between LNP products and autoimmune symptoms.¹²

While LNP specific characteristics are not centrally germane to this article, it is worthwhile to mention publication of a recent draft guidance document titled Drug Products, Including Biological Products, that Contain Nanomaterials Guidance for Industry. This draft guidance document contains a significant amount of information directly relevant to the products that contain LNPs.¹³

Lipid components in cancer vaccines, prophylactic vaccines, and genome editing materials could be theoretically considered as either critical components of the drug product (DP), biological raw materials, starting materials, or novel excipients.

Why is it important that we develop a consensus as to the function of lipids in LNP complexes used in therapeutic and prophylactic vaccines?

It is important to reiterate that from a regulatory requirement perspective, the extent of material characterization and release depends largely on how lipids are categorized case by case. For example, the FDA’s expectation of manufacturers to assess the quality of materials in DS, excipients, or critical components of the DP is higher than for consumable material, raw materials, and material components used for the final drug product manufacturing. While the former material is expected to be assessed for identity, purity, potency, activity, and safety, the latter can be generally qualified based on material qualification and assessment of limited quality attributes as summarized in the manufacturer’s COA.

Important Questions To Help Us Define Lipid Nanoparticles

To this day, regulatory bodies struggle to fit lipid nanoparticles into a category. Are they active ingredients? Are they excipients? Are they critical components? Let’s look at the parts of a drug and see what shakes out.

What is an active ingredient or drug substance?

According to 21 CFR 210.3(b),¹⁴ DS is an active ingredient in any component of the drug product intended to furnish pharmaceutical activity or affect the structure and function of the human body. Based on this definition, because lipids function to stabilize and enhance delivery of the major active ingredient of the DP, namely mRNA, by a mechanism that involves modifying target cells ex vivo or in vivo, these materials should not be considered as drug substance, even though without lipids the major active ingredient of the presumed DP is or becomes ineffective.   

What are new excipients?

According to a final guidance document, novel excipients are any inactive ingredients that are intentionally added to therapeutic product that:

• are not intended to exert therapeutic effects at the intended dosage, although they may act to improve product delivery and
• are not fully qualified by existing safety data.¹⁵

According to this definition, lipids in LNPs could function as novel excipients. If so, novel excipients are required to undergo prior safety assessment¹⁵ or at least follow the novel excipient review program.¹⁶

What are biological raw materials?

As defined by guidance documents from the FDA¹⁷ and Health Canada¹⁸, raw material is a general term used to denote starting materials, reagents, and solvents intended for use in the production of intermediates or active pharmaceutical ingredients (APIs). In general, these materials are consumed during manufacturing and are not found in the final drug product. Trace quantities of raw materials may be detected in the final drug product as process-related impurities. According to this definition, lipids cannot and should not be considered as raw materials as they are intentionally added to the product’s active ingredient.¹⁴

What are starting materials or starting source materials?

Starting materials are not very well defined in the FDA guidance document but generally contain active ingredients, as defined in reference 17: “For purposes of this document, material from a biological source which is intended to be used in the manufacture of a biological product and from which the active ingredient is derived either directly (e.g., plasma derivatives, ascetic fluid, bovine lung) or indirectly (e.g., cell substrate, host/vector production cells, eggs, viral strains). Based on this definition, lipids, which are derived from a biological starting source such as plants or eggs or manufactured synthetically, may be considered starting materials; however, because the active ingredients of therapeutic and prophylactic vaccines are not derived from lipids or lipid sources, then this definition may not be suitable from a regulatory perspective.

What is the critical component of the drug product?

FDA presumably refers to lipids as critical components of the genome editing (GE) products at minimum.^{19, 20} For example, when the GE components are expressed in vivo by directly administered plasmids or vectors, the plasmid or vector in its final formulation encoding the GE component is considered the DP. If used to modify cells ex vivo, the GE components are considered critical components for the manufacture of the final product because, without these components, the resulting drug product would not have the same pharmacological activity.²⁰

Although FDA has not explicitly mentioned lipid components in this particular draft guidance document, based on this statement it is reasonable to conclude that lipids used for LNP manufacturing of DS/DP for genome editing products could be considered as critical components of DP at minimum. Furthermore, lipids could be reasonably considered critical components because lipids are present in the final formulated product. Furthermore, without lipids, the resulting finished product would not have the same pharmacological activity.

Classification Aside, Impurity Mitigation Is Critical

Although we do not have a clear consensus from regulatory authorities, lipids used for manufacturing prophylactic and therapeutic vaccines could be considered as either novel excipients or critical components of the drug product based on intended use. For example, in Europe, lipids used for formulation of SARs-Cov2 vaccine were considered to be non-compendial novel excipients. In contrast, lipids in Moderna vaccines were reviewed as starting materials.²

Regardless of the exact terminology and classification used by sponsors, since the major impurities in the materials used to manufacture in vivo genome editing product or ex vivo edited cells or vaccines could contribute significant impurities to the final drug product, which cannot be mitigated by downstream processing including sterile filtration, the quality of material must be assessed more comprehensively as compared to other ancillary raw materials used further upstream.

For example, endotoxin is a major contaminant that cannot be effectively filtered out in downstream processes. As such, the level of endotoxin in the lipids should be monitored and controlled. Accordingly, the quality of lipids must be controlled by establishing appropriate specifications that include test method, procedure, and acceptance criteria for sterility, identity, purity, and activity in a phase-appropriate manner.¹⁹ It is important to emphasize that this expectation of quality assessment is markedly different from the testing requirements for the raw materials, which are consumed during manufacturing.

Table 3 below summarizes suggested tests for assessing the quality of the lipid components to be used for LNP formulation.

In general, the required specification for lipids used as either critical materials or novel excipients depends on many factors, including intended use, the source of material, manufacturing process, and risk assessment.

The rationale for including extensive testing of lipids is based on the following considerations:

• Lipids represent a major component of the final drug product.
• Contamination in lipid materials, which cannot be mitigated by downstream processing, could potentially pose a significant risk to healthy individuals (prophylactic use) and patient population (therapeutic use).
• A large batch size of lipids typically used could potentially become the source of contamination and/or impurities that could pose a significant risk to a large number of healthy individuals or patients who are treated when used in an off-the-shelf manner.
• The lipids discussed here have arguably limited prior-use histories.

Examples of relevant material quality attributes for lipid components include a measure of identity, purity, impurities, and safety. Specifications provided are for illustrative purposes and should include tests, test methods, and acceptance criteria. In general, acceptance criteria are informed by prior historical data and acceptable limits that ensure the quality of the final product.

It is important to note while cationic synthetic lipids and pegylated lipids require well-defined specification for release, the quality of DSPC and cholesterol could potentially be assessed using USP monographs.^{21, 22} However, USP monographs may not provide sufficient evidence of safety and potency.

Overall, based on publicly available information, it is not clear that prototype lipid identified below as an example, sourced from major manufacturers, are adequately characterized in view of potential risk of potential impurities that cannot be mitigated by current downstream processing.

Table 3: Specification examples

Product/manufacturer	Source	Identity	Purity	Sterility/bioburden control including bacteria and fungi	Endotoxin	Impurities (product- and process-related)
Cationic Lipid ALC-0315, (BionTech/Pfizer), DLin-MC3-DMA (Alnylam), SM-102 (Moderna)	Synthetic	Specification: Molecular weight mass spectroscopy acceptance Criteria: Identical	Specification: Percent purity HPLC Acceptance Criteria: >98%	Specification: Culture based growth or non-compendial methods Acceptance Criteria: No growth or for bioburden control NMT X CFU/mg	Specification: LAL chromogenic assay NMT X EU/mg	Specification: Product related impurities include tests to measure different forms of lipid synthesized. Process related impurities include, for example, any solvents used during synthesis.

Proposed extended test of lipid materials. Specifications include tests, test method, and acceptance criteria. NMT: Not More Than

Conclusion

Today, with very few commercial products containing LNPs, we have accumulated a body of evidence about the therapeutic application of LNP complexes, which tells us that larger and more frequent administration of LNPs is required to achieve therapeutic effects.

Based on available published data accumulated in preclinical and human trials, we can hypothesize about the factors contributing to the observed toxicity, which is likely associated with the particle size, composition of lipid particles, and exact nature of cargo being delivered.

It is also well understood that nanoparticles containing nucleic acid agents are recognized as foreign materials by the body and stimulate innate immunity, which in turn impacts adaptive immunity. Another aspect that requires further attention is how lipids’ material quality is currently assessed as either novel excipients or critical components of the DP.

In summary, LNP technologies have advanced the field of infectious disease control, cancer vaccines, and treatment of genetic diseases. Inherent in the action of LNP complexes is their ability to enter the cells and deliver their cargo to target cells using a well-understood pathway, which shares characteristics of agents that elicit innate and adaptive cell-mediated responses. If these responses are not appropriately controlled, they could potentially lead to severe adverse events.

In the short term, we must minimize the dose of LNPs by conducting careful studies to better understand the maximal acceptable threshold for toxicity of LNP formulations using dose-finding studies in preclinical models and Phase 1 human trials. It is also critical that we continue to improve the quality of critical materials used for LNP manufacturing. Specifically, I argue that the quality of lipid components used for manufacturing should not be tested as another raw material based on COA from manufacturers but, rather, as critical components of DP or novel excipients used for LNP manufacturing. I recommend this approach because lipids are present in the final drug product and play a major role in stabilizing the active ingredient of the final product, the mRNA component; any contaminant or impurities introduced by lipid materials may potentially pose a significant risk to the recipients in the context of intended use, dosage, and manufacturing processes.

In the future, it will be most critical that we continue research toward the development of safer LNP formulations factoring in not only the chemical composition and quality of lipids used for LNP formulation but also exploring ways to improve the quality of mRNA components used in manufacturing and ways to deliver nucleic acid more efficiently to target cell/tissue in vivo.¹⁴

In addition, it is my opinion that different components of lipids should be considered at minimum as critical components of the drug product and/or novel excipients on a case-by-case analysis and should be tested and released based on adequate predetermined specifications discussed in this article. Furthermore, the development of monographs for lipid components as novel excipients will likely accelerate the development of novel LNPs for vaccine and cell and gene therapy products. A lack of clear consensus in the field for assessing the quality of lipids in LNP formulation based on various intended uses necessitates a call to action for regulatory bodies, including approving authorities and pharmacopeias, to join efforts with industry and develop monographs for novel excipients, as this will support innovation and bringing new drug formulations to market. This is a critical step toward bringing more emerging therapies to patients.

References:

1. Assessment report on the claim of new active substance (NAS) status of 5’capped mRNA encoding full length SRAS-CoV-2 Spike protein contained in COVID-19 mRNA Vaccine BioNTech. EMEA/H/C/005735/RR (https://voorwaarheid.nl/wp-content/uploads/2022/12/Rapporteurs-Rolling-Review-Report-New-Active-Substance-Status-COVID-19-mRNA-Vaccine-BioNTec.pdf).
2. BLA Clinical Review Memorandum, SPIKEVAX STN: 125752 Proper Name: COVID-19 Vaccine, mRNA, Tradename: SPIKEVAX, Manufacturer: Moderna Tx Inc. (https://www.fda.gov/media/156342/download), (https://www.fda.gov/vaccines-blood-biologics/spikevax).
3. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032477/pdf/main.pdf.
4. Clinical advances and ongoing trials of mRNA vaccines for cancer treatment (https://www.thelancet.com/action/showPdf?pii=S1470-2045%2822%2900372-2).
5. Verve data captured from company’s website (https://www.vervetx.com/our-programs/verve-101-102)
6. Reflections on Alnylam (https://www.nature.com/articles/s41587-022-01304-3.pdf)
7. Center For Drug Evaluation AND Research Application Number: 215515Orig1s000 Summary Review (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2022/215515Orig1s000SumR.pdf)
8. Small Interfering RNA (siRNA) Therapy (https://www.ncbi.nlm.nih.gov/books/NBK580472/)
9. Center for Drug Evaluation and Research Application Number:210922Orig1s000. Product Quality Review(S) OPQ-XOPQ-TEM-0001v04 Page 1 of 3 Effective Date: 14 February 2017 Quality Assessment NDA 210922. ONPATTRO (patisiran) Lipid Complex Injection (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210922Orig1s000ChemR.pdf)
10. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability - PubMed (nih.gov)
11. A perspective of lipid nanoparticles for RNA delivery (https://onlinelibrary.wiley.com/doi/full/10.1002/EXP.20230147)
12. Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics (https://www.nature.com/articles/s12276-023-01086-x.pdf)
13. Drug Products, Including Biological Products, that Contain Nanomaterials Guidance for Industry Drug Products, Including Biological Products, that Contain Nanomaterials Guidance for Industry.
14. Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients; Guidance for Industry, September 2016. Available at https://www.fda.gov/media/71518/download
15. Guidance for Industry Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (https://www.fda.gov/media/72260/download)
16. Pilot Program for the Review of Innovation and Modernization of Excipients (PRIME) https://www.fda.gov/drugs/development-approval-process-drugs/pilot-program-review-innovation-and-modernization-excipients-prime
17. Chemistry, Manufacturing, and Controls Changes to an approved Applications: Certain Biological Products (https://www.fda.gov/media/109615/download)
18. Health Canada Guidance Document: Post – Notice of Compliance (NOC) Changes: Quality Document, Government of Canada, September 2009. Available at https://www.canada.ca/en/health-canada/services/drugs-health-products/drug-products/applications-submissions/guidance-documents/post-notice-compliance-changes/quality-document.html
19. Gene therapy and genome editing guidance document (https://www.fda.gov/media/156894/download)
20. Human Gene Therapy Products Incorporating Human Genome Editing Guidance for Industry (https://www.fda.gov/media/113760/download)
21. DSPC Monograph: https://doi.usp.org/USPNF/USPNF_M16917_10101_01.html
22. USP Cholesterol Monograph: (https://doi.usp.org/USPNF/USPNF_M17220_03_01.html).

About The Author:

Mo Heidaran, Ph.D., is currently chief regulatory scientist at Cellx Inc. He previously worked as head of translational and regulatory strategy at GCTx (GC Therapeutics). He is an expert in the development of cell and gene therapies with more than nine years of experience at the FDA’s Center for Biologics Evaluation and Research, more than three years at Parexel International as vice president of technical, and 15 years as R&D director in the biotech industry working in companies like Celgene, BD, and Johnson & Johnson. His career began in 1987 at the National Institutes of Health as a senior staff scientist. He has more than 30 years of in-depth expertise in all aspects of regulatory compliance, clinical trial logistics and operation, product, process development, process optimization, and scalable CGMP-compliant biomanufacturing of cell and gene therapeutic products.