Friedrich von Wintzingerode, Ph.D. - QC Lead iNeST Genentech, Inc. A member of the Roche Group
Introduction
Advanced therapy medicinal products (ATMPs) also referred to as Cell and Gene Therapy products (CGT) are a new class of drugs which is fundamentally different from traditional drugs like therapeutic antibodies or small molecule drugs. ATMP manufacturing covers an extremely wide range of different process technologies from cell-free production of mRNA therapeutics to ex-vivo engineering of human cells for autologous or allogeneic cell therapies to viral vector induced in-vivo gene therapies. One of the most significant differences between traditional drugs and ATMPs was the advent of individualized therapeutics, which are “make-to-order” medicines produced for a particular patient. Each batch of an individualized product is manufactured for a specific patient. Since each batch has unique characteristics that are dependent upon the patient sample characteristics and production process, the process largely defines the product. There are several examples of individualized therapeutics in development or on the market. One of these are autologous T-cell products like chimeric antigen receptor T-cells (CAR-T). Another example are individualized neoantigen-specific therapies (iNeST), which can have a final drug product that is mRNA or DNA. The following paper describes a cell-free iNeST-mRNA process and the microbial controls in place to ensure microbially safe manufacturing for ongoing clinical trials.
iNeST mRNA Project Introduction
The iNeST mRNA immunotherapy project is a partnership between Roche-Genentech and BioNTech. Each iNeST drug product is manufactured on a per-patient basis and consists of an mRNA encoding neoepitopes in a nanoparticulate (Lipoplex) formulation.
The end-to-end process is outlined in Figure 1 and consists of an upstream target identification and selection process where potentially immunogenic neoepitopes are identified and a downstream process where each drug substance and drug product is manufactured.
Upstream Manufacturing
Upstream manufacturing for iNeST mRNA starts with next-generation sequencing (NGS) of the patient specific tumor biopsy and blood samples followed by a target selection process) that generates an electronic data file listing proposed neoepitopes. The selected neoepitopes are used to design the sequence of the patient-specific cassette of the DNA template. This concept is fundamentally different from traditional drug manufacturing where upstream manufacturing results in a physical process intermediate (e.g., harvested cells after CHO cell cultivation for monoclonal antibodies).
Downstream Manufacturing
Downstream manufacturing starts with synthesis of the DNA template followed by in-vitro transcription, purification and encapsulation of resulting mRNA in lipoplexes. The mRNA lipoplexes are then aseptically filled into vials and administered to the respective patient via intravenous injection.
Points to Consider for Defining a Microbial Control Strategy for iNeST mRNA
The development of a microbial control strategy for ATMPs requires an understanding of the developing regulatory environment. Major health authorities like EMA and FDA have published several ATMP specific guidelines 1,2,4,5. So far health authorities have not published guidelines specific for microbiological topics for ATMPs yet. However, EMA has stated that the following microbiological EP monographs should be considered for ATMPs, where relevant5:
- Ph.Eur. 2.6.21 Nucleic Acid Amplifi cation Techniques
- Ph.Eur: 2.6.27 Microbiological control of cellular products
- Ph.Eur: 2.6.1 Sterility
- Ph.Eur: 5.1.6 Alternative methods for control of microbiological quality
- Ph.Eur. 2.6.7 Mycoplasmas
- Ph.Eur. 2.6.14 Bacterial endotoxin testing
Within the regulatory framework the microbial control strategy for iNeST mRNA was developed based on the unique risk profile of individualized therapeutics, key characteristics of the downstream process, and the tight manufacturing turnaround time (TAT) requirements for individualized therapeutics.
Risk Profile
Compared to traditional drugs where each batch is produced for hundreds or even thousands of patients each individualized therapeutic manufacturing run creates a unique (patient specific) Drug Substance and Drug Product batch. Therefore, the impact of a microbial contamination is lower for individualized therapeutics because each product batch is only affecting one patient.
Key Characteristics of the Downstream Process
In contrast to traditional drug manufacturing processes like CHO cell fermentation and protein purification iNeST mRNA manufacturing is a well-defined in-vitro process with no practical separation between DS and DP manufacturing. iNeST mRNA has the following key characteristics
- All product contacting material is single-use
- DS manufacturing is highly automated
- Short manufacturing time
- Completely cell-free
- Absence of complex raw materials
- Encapsulation of mRNA in lipoplexes happens under aseptic conditions using sterile components since mRNA lipoplexes cannot be terminally sterilized
Turnaround Time (TAT) Requirements
Individualized therapies are subject to tight TAT requirements to ensure timely drug administrations to patients. At first glance, this makes rapid micro methods (RMM) an attractive method candidate due to their short time-to-result (TTR) compared to traditional micro methods.
However, it would be an oversimplification to conclude that individualized therapeutics automatically require RMM. Rather, the selection of micro methods for individualized therapeutics must consider all of the following criteria:
- Scientifically sound - The method must be properly validated and follow industry standards.
- Short hands on time - Short, simple workflows decrease the risk of errors during method execution.
- Reliable - The method should have a low false positive rate and low system suitability failures to avoid time consuming investigations.
- Flexible execution- It is preferable for micro methods to be executable 6-7 days per week to avoid delays to QC testing and batch release.
Compendial micro methods meet all of the aforementioned criteria which makes them methods of choice for individualized therapeutics as long as method TTR does not negatively impact manufacturing TAT. For iNeST mRNA a detailed mapping of the GMP manufacturing process identified DP release testing as TAT critical whereas DS and in[1]process testing are not TAT critical (see Figure 2). Consequently, a rapid sterility test was implemented for DP release because the 14 day TTR of compendial sterility testing would have increased manufacturing TAT whereas compendial bioburden testing was implemented for DS and in-process testing (see The iNeST mRNA microbial control strategy).
The iNeST mRNA Microbial Control Strategy
Taking the risk profile of individualized therapeutics, key characteristics of the downstream process, and the tight TAT requirements into consideration the following microbial control strategy was defined for iNeST mRNA manufacturing.
Control of raw materials
All raw materials are of animal-free origin from qualified vendors. Raw materials used for DS manufacturing (enzymes, nucleotides, buffers) are tested for endotoxins and/or bioburden. Raw materials used for DP manufacturing are added to the process as sterile solutions.
In-process testing
The simplicity of the iNeST mRNA manufacturing process significantly reduces the risk of microbial contaminations and enables a lean in-process testing strategy, which is focused on critical process steps as outlined in Figure 3. Bioburden samples are taken after RNA purification prior to the first 0.22 µm filtration step in the process andprior to 0.22 µm sterile filtration of the diluted and conditioned RNA Drug Substance. A pre sterile filtration bioburden sample is also taken from solutions which are added to the Drug Product manufacturing process. All bioburden samples are tested using compendial membrane filtration which has proven to be a reliable and robust testing approach with no negative impact on manufacturing TAT.
Drug product release testing
The iNeST mRNA DP is an intravenously administered drug and tested for sterility and endotoxins. The Celsis™ method is used as a rapid sterility test. A TTR of 7 days was validated according to the principles outlined in Ph. Eur. 5.1.6., USP, and PDA Technical Report No. 33. This is a significant improvement for manufacturing TAT compared to the 14 days TTR of the compendial sterility test. Endotoxin testing for DP release is performed using the compendial LAL assay per EP 2.6.14/USP<85>.
Conclusion
The iNeST mRNA individualized therapy requires a microbial control strategy which takes the risk profile of individualized therapeutics, key characteristics of the process, and the tight TAT requirements for individualized therapeutics into consideration. Manufacturing TAT is the key factor for test method selection which led to the implementation of rapid sterility testing for DP release. Bioburden and endotoxin testing is performed using compendial methods as this has proven to be a robust and reliable testing approach that meets TAT requirements.
Acknowledgements
The author would like to thank Manuel Mercier, Ben Tran, Chris Petry, and Christoph Kreitner for their contributions to the development of the microbial control strategy for iNeST mRNA and Eva Radmacher and Jonas van den Berg for the validation of the Celsis rapid sterility test.
References
- EudraLex - Volume 4, part IV “Guidelines on GMP specific to Advanced Therapy Medicinal Products”
- EMA/CAT/80183/2014 “Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products”
- Draft guideline EMA/CAT/852602/2018 “Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials”
- FDA 2020 guideline “Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)”
- https://www.ema.europa.eu/en/human-regulatory/research-development/advancedtherapies/guidelines-relevant-advanced-therapy-medicinal-products
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