Major CMC Commercialization Gaps for Gene Therapy Products Manufactured for a Diverse Pediatric Subpopulation(s)

The development of cell and gene therapy products to address serious and life-threatening conditions that affect the pediatric population is growing exponentially. Although much is known about the Chemistry and Manufacturing Control (CMC) considerations for the adult population, the challenges of manufacturing complex products for younger populations are not yet fully understood.

To manufacture cell and gene therapy products for pediatric subpopulations, special considerations need to be taken into account and there is a good reason to conclude that the relative risk due to drugs increases inversely by age. For example:

1. Risk to patients for the same drug product is generally higher for young children than for adolescents and young adults;
2. We have generally more knowledge of a drug’s effects in the adult patient population;
3. Infants are generally harder to treat and manage. For example, it may be harder to control immune response manifestations, such as fever, in infants;
4. The pediatric population is comprised of patients with a broad range of age, weight and organ development. Infants have organ development patterns that do not necessarily correlate linearly with increase in body size and weight.

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According to the U.S. Food and Drug Administration (FDA) and labeling regulations found in Code of Federal Regulations (CFR) Title 21, 201.57(c)(9)(iv), pediatric patients range in age from 0-16, and the age range depends on context. Additionally, children are defined as persons who have not attained the legal age for consent to treatment (21 CFR 50.3(o)). The pediatric population as defined in the FDA regulation, however, encompasses a wide range of patients that have very heterogenous physical characteristics, organ development, and other patho-physiological characteristics, which can make developing drug products for this population extremely challenging.

Pediatric Population Age Range and Organ Development

Although there are no comprehensive studies performed to delineate the state of organ development in various pediatric subpopulations, available data strongly suggests that the development of organs in pediatric subpopulations does not necessarily scale linearly with age or body weight. For example, the majority of brain development occurs early in life and plateaus soon after adolescence.^1,2 This pattern of early development is remarkably different than that shown for other organs, including liver and lung, which correlate more linearly with age and weight. As a result, identifying appropriate dosing for different pediatric subpopulations based on total body weight and age can be extremely difficult, particularly for certain routes of administration.

In addition, due to benefit/risk considerations, a general principle for cell and gene therapy trials is that initiation of a First-in-Human (FIH) trial depends on identification of a safe starting dose of the experimental drug that is expected to have efficacy in the target population. For pediatric clinical trials, this expectation for efficacy is known as prospect of direct benefit. As per 21 CFR 50, Subpart D, evidence of a prospect of direct benefit is required to administer any product to pediatric patients that is associated with more than a minor increase over minimal risk. For example, the starting dose of cell therapy products may be expressed as the total of X number of cells, which is determined based on a predetermined volume of cell suspension having strength or concentration of Y number of cells per unit volume. Typically, the number of cells given to a patient is expressed in terms of number of cells per kilogram only, without considering age and other variables. As a result, the volume of the product infused could become a rate-limiting factor in certain patient populations.

Table I below summarizes some of the important considerations regarding cell and gene therapy. Dose, dosage form, strength, dose regimen and infusion rate are important parameters to be considered for determining the exact Target Product Profile (TPP).

Volume Consideration for Pediatric Subpopulations

Because cell and gene therapy products are constituted in a certain unit, volume administration of an effective dose in a certain patient subpopulation, such as premature babies, may be very challenging. This is particularly relevant if the route of administration could also further complicate this matter. For this reason, manufacturers are encouraged to not define an effective dose of a drug product based on what is a practical volume to administer to a certain target population based on the adult formulations. Accordingly, it is essential that a final drug product is formulated at a volume that maximizes dose and efficacy for the target pediatric subpopulations. In addition, to define dose and strength for pediatric subpopulations, the manufacturers should also consider other variables, such as infusion rate, which should be optimized and managed to mitigate risk. Also, since in the administration procedure methodology can affect the efficacy and safety profile of the product in the target subpopulation, for any given route of administration, it is essential for the manufacturers to develop strategies to maximize effective dose in the context of safe infusion volume and rate. This strategy may require patient-specific approaches to the drug formulations and post-thaw wash and reconstitution for cell therapy products and volume reduction for viral product at the manufacturing facility or at the clinical site.

To better frame special CMC consideration for pediatric populations, it is important to touch upon some basic definitions of safety and purity in the Code of Federal Regulations (CFR).

• “Safety" is defined as the relative freedom from harmful effects to persons affected, directly or indirectly, by a product when prudently administered, taking into considerations the character of the product in relations to the condition of the recipients at the time (21 CFR 600.3(p)).
• As such it is important to emphasize that “product safety” is established in relationship to the condition of recipients (age, health conditions, etc.). A safe product for an adult population may not be necessarily safe for a pediatric population in view of their age, developmental stage and other conditions.
• “Purity” is carefully defined as “Product shall be free of extraneous materials except that which is unavoidable in the manufacturing process” (21 CFR 610.13). Purity also means relative freedom from extraneous matter in a finished product whether or not harmful to the recipient or deleterious to the product (21 CFR 600.3(r)). This means that the final drug product must be essentially free of any extraneous materials that are avoidable whether or not they have proven deleterious effects on the patient population. For this reason, it is particularly important to emphasize the importance of product purity in the context of pediatric subpopulations, which are shown to be sensitive to some particular impurities, such as visible particulates and endotoxins.

The overall CMC considerations for various pediatric subpopulations are highly complex and include the following key points:

• Product Dose, Strength and Volume considerations
• Product Purity Profile 
  • Inactive Ingredients (cryopreservative and stabilizers) 
  • Particulates in Products 
  • Endotoxin 
• Route of Administration 
  • Intrathecal
• Injection to Spinal Canal
• Subarachnoid Fluid
• Cerebrospinal Fluid
  • Intravenous 
  • Intraperitoneal
• Site of Administration 
  • Ocular 
  • Heart 
  • Systemic
• Indications 
  • Cancer versus Autoimmune Conditions Therapy
• Patient Populations 
  • Pediatric (age range)
• Concomitant Therapies
• Delivery Devices

In this article I will attempt to touch upon some key considerations as outlined below.

Product Administration-Devices Used for Delivery of Cell and Gene Therapy Products

Generally, devices used to deliver cell and gene therapy products are not cleared specifically for delivery of very complex therapeutics including cell and gene therapy products. That is, even if the device is cleared for a different delivery indication (e.g., a catheter that is indicated for intravascular delivery of radiocontrast), it would be considered to be an investigational device for a non-cleared indication (e.g., intravascular delivery of a gene therapy product). For this reason, the device compatibility study should be performed with the actual drug product and should mimic the condition of use and patient characteristics intended to be treated. It is important to keep in mind that for any given device, there are some differences in biocompatibility of the device used in a wide range of pediatric patient subpopulations, factors which should be considered in choosing the actual delivery device and route of administration.

Product Administration-Cerebrospinal Fluid (CSF) Dosing Considerations in Children and Adults

To highlight the importance of product administration, let’s consider a hypothetical case of administering a dose of viral products to various pediatric subpopulations, ranging from 0-16 years of age. For Intrathecal (IT) administration of this product into Cerebral Spinal Fluid (CSF), it is important that manufacturers have reliable data about the volume of CSF in different pediatric subpopulations. For example, reference textbooks state that preterm and full-term infants have a much greater CSF volume relative to their weight (11 and 15 ml kg−1, respectively) than a child or adult (4 and 2 ml kg−1, respectively).³

However, other recent publications state the amount of spinal CSF in neonates can be estimated as 2 ml/kg in both term and formerly preterm neonates.⁴

In determining the most optimal formulation of the drug product for each pediatric subpopulation, manufacturers should consider the volume of the injectate that would be safe to deliver, which is based on empirical evidence of historical data published in peer reviewed journals. Nonetheless, it is important to point out that current available data suggest that CSF volume is different for distinct pediatric subpopulations, CSF volume is not proportional to weight, and published results are not always consistent.

Cell and Gene Therapy Product Formulation: Considerations for Pediatric Populations

For cell and gene therapy products, as shown in Table 2 below, the final drug product contains inactive ingredients, such as cryopreservatives (cell therapy products) and stabilizers (gene therapy products), that can potentially pose a safety risk to certain pediatric subpopulations. To mitigate risks associated with cryopreservatives and stabilizers, cellular products can be thawed and washed at the clinical site, or such agents can be diluted.

Overall it is reasonable to suggest that the risk associated with the presence of inactive ingredients in the final drug product may be higher in pediatric populations and should be mitigated appropriately.

Product Quality Standards: Particulates in the Final Drug Product-Safety Reports in Neonates

The presence of foreign visible or sub-visible particulate matter in parenteral formulations has been one of the most common reasons for product recalls. From 2008 to 2012, the FDA reported that 22% of recalls for sterile injectable drugs were caused by the presence of visible particles.⁵ The presence of particulates poses safety concerns for cell and gene therapy products, as they are characterized as impurities in the final drug products. There are three types of particulates:

• Inherent (i.e. cell clump or virus aggregate)
• Intrinsic (i.e. particulates coming for product contact surfaces)
• Extrinsic (i.e. hair, fiber coming from manufacturing environments)

Each type of particulate can be further characterized based on size. For visible particles, the size range is typically more than 100 µM, while for subvisible particles, the size range is generally below 100 µM.

In a publication by Dominic et al.,⁶ a chart is provided that summarizes the risk for the presence of particulate in the final drug product which is dependent on patient type with pediatric patients having the highest risk and infusion route with intravenous (IV) and intrathecal (IT) being the most critical. For this reason, the sponsors of experimental cell and gene therapy products are highly encouraged by FDA to develop strategies to mitigate risk associated with the presence of particulates in the final drug product. This approach of identifying potentially harmful particulates in the final product is particularly complicated for cell-based products, which consist of inherent particulates. The measurement of intrinsic and extrinsic particulates is also very technically difficult.

Based on lesson learned from regulatory considerations for control of visible and subvisible particulates for parenteral drug and biological products including monoclonal antibodies and recombinant proteins⁷ combined with our expanding knowledge of the cell and gene therapy products key recommendations for managing particulates in the final drug product for pediatric population would involve conducting a detailed risk assessment. This approach involves:

• Identifying types of particulates and potential source of particulates in the final drug product
• Reducing sources of particulates in the final drug product
• Controlling strategy to minimize level of particulates in the final product include but are not limited to:
  • Control manufacturing environment
  • Control introduction of particulates by components especially containers with direct contact with product
  • Choose containers that allows for visual inspection
  • Perform visual inspection for presence of visible particles (due to aggregation)
• Develop tests for measuring and monitoring visible and subvisible particles in cell and gene therapy products

Product Quality Standards: Endotoxin Level Control Strategy and Testing

Endotoxin is one of the most important bacterial components contributing to the inflammatory process. Endotoxin acts on neutrophils, platelets and complement to produce, both directly and through mast cell degranulation, vasoactive amines that cause hypotension.

To assess the safety of drug products, the compendial Bacterial Endotoxin Test (BET) measures the levels of resident endotoxins against a product specific, dose-dependent, route of administration- dependent and time of administration-dependent calculation called the endotoxin limit.⁸

If the target patient population is adults, it is generally assumed that the average adult in the US weighs 70kg. However, the average adult in Japan is assumed to weigh 60kg. If the target patient population is a spectrum of patients, the maximum dose/kg/hr is generally the largest dose given to the smallest patient in the target group. Pediatric population exposure to endotoxin is expressed in terms of maximum dose/kg/hr.

According to USP and guidance documents, manufacturers are required to perform pyrogenicity tests for biological products. If it cannot be performed in rabbits, then alternative methods are acceptable, as long as they are fully validated, like the Limulus Amoebocyte Lysate (LAL) test.⁹

In addition according to the Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs)-2008 and recently published CMC information for human gene therapy investigational new drug application¹⁰ “For any parenteral drug, except those administered intrathecally, we recommend that the upper limit of acceptance criterion for endotoxin be 5 EU/kg body weight/hour. For intrathecally-administered drugs, we recommend an upper limit of acceptance criterion of 0.2 EU/kg body weight/ hour. You should describe in your IND the pyrogenicity/endotoxin testing you conduct, and your acceptance criterion for release”.

However, the major issue associated with LAL is the presence of agents in the test sample matrix that interfere with endotoxin detection and are common in biological products. The inhibition or masking event can theoretically be considered both irreversible or reversible, the latter of which is more easily addressable, while the former further complicates the interpretation of the product safety. The reversible inhibition of endotoxin detection by the presence of the biological matrix is often addressed by pretreatment of sample or dilution of the sample that leads to the recovery of endotoxin. In contrast to the reversible component of the endotoxin detection there is a component of inhibition which is irreversible. Accordingly, the irreversible component of endotoxins recovery by unknown “factors” present in the final drug product could potentially pose significant safety issues particularly for pediatric populations.

This phenomenon is described in detail in a recently published article on Low Endotoxin Recovery (LER) and potential solutions entitled “PDA Technical Report on Low Endotoxin Recovery: Implications to the Industry”. Since first reported by Chen, J., and Vinther, A. in 2013,¹¹ the phenomenon of LER has been broadly observed in certain matrices commonly used for biologic formulations and certain therapeutic proteins. LER is defined as the inability to recover >50% activity over time when endotoxin is added to an undiluted product. According to this review “LER is a temperature-and time dependent process, which usually does not occur immediately but after several hours to several days”. The authors emphasize the importance of sample preparation as the most critical aspect of performing an endotoxin test which is the most effective strategy to mitigate risk associated with the endotoxin masking found in cell and gene therapy products.¹²

Other confounding factors when measuring endotoxin exposure levels would be whether or not the endotoxin limits should apply to accumulative patient exposure to bacterial components found in other medicinal product administered to patient concomitantly.

For example, patient X receive an allogenic cellular product and immunosuppressive drug along with a checkpoint inhibitors at the same time. What should be the endotoxin limit?

The 5 EU/kg/hr should be the limit for all drugs infused to the patient at the same time or this limit should be restricted to the allogenic cellular product only. In theory it is important to implement a strategy that minimizes patient exposure to endotoxins by adhering to the recommended limit of 5 EU/kg/hr for intravenous infusion but it is also understood that in some cases where combination therapies are used this approach could be sometime impractical.

The schematic below offers a few points to consider to identify and measure true concentration of endotoxin, as well as good manufacturing practices, which can be used to reduce effective concertation endotoxin in the final drug products.

Endotoxin Risk Mitigation for Pediatric Subpopulations

The control strategy to minimize introduction of endotoxin into the final drug product include

1. control and reduce endotoxin level of all components (ancillary materials)
2. control of manufacturing environment to eliminate the likelihood of any introduction of endotoxins from the environment
3. control of endotoxin levels in the starting materials and
4. consider validation of endotoxin test early on- factoring in possible endotoxin masking by biological factors in the product-It is not straightforward.

In summary, CMC considerations for pediatric subpopulations remains to be very complex. However, key issues and possible risk mitigation strategies involve, but are not limited to, the following (see Figure 1):

 

• Dose issue: One product with one strength for all pediatric populations.
• Risk mitigation strategy: Dose for pediatric patient populations should be optimized based on experimental and scientific evidence. Dose must be adjusted for target patient populations.
• Volume issue strategy: To achieve effective doses, large volumes may have to be administered, which is not practical.
• Risk mitigation: Optimize drug concentration – i.e., cell number per mL or virus particles per mL, to maximize number of cells or virus particles delivered per unit volume of the drug.
• Particulate issue strategy: Some products may contain cell aggregate clumps or virus aggregate.
• Risk mitigation: Identify source of aggregates and particulates, minimize particulates in the drug product and implement a method for measurement of particles in the final drug product, identify meaningful release specification.
• Endotoxin levels issues: Levels may exceed the recommended EU/Kg/hr.
• Risk mitigation: Identify the source of the endotoxins in the final drug products (in starting material or ancillary material or environment); if not successful, increase infusion time or perform multiple infusions at distinct time points.

Finally, it is also important to keep in mind that infants and children are not miniature adults. As a result, the development of highly effective cellular and gene therapy products for this very heterogenous subpopulation may require further patient specific optimization / customization. In such a case, the product dose and strength should be ideally optimized for different pediatric patient subpopulations based on age, developmental characteristics, weight, and site/route of administration.

Acknowledgement

The author would like to thank Dr. Steve Winitsky for his contributions to this article.

References

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3. Lups S, Haan AMFH, Bailey P. The Cerebrospinal Fluid, 1st Edn. 350 pp. Amsterdam: Elsevier, 1954 Suresh S, Polaner DM, Coté CJ. In: Coté CJ, Lerman J, Anderson BJ, eds. Regional Anesthesia, a Practice of Anesthesia for Infants and Children, 5th Edn. Philadelphia: Elsevier Saunders, 2013; 835–79
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9. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/pyrogen- and-endotoxins-testing-questions-and-answers
10. https://www.fda.gov/media/113760/download
11. Chen J, Vinther A. Low endotoxin recovery in common biologics products. Presented at the PDA Annual Meeting, Orlando, FL. April 15-17, 2013
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