In this guest editorial, BioPhorum summarizes its latest publication that aims to reach a consensus on test attributes and release requirements for plasmids and the bacterial master cell banks (MCBs) used to produce them.
Plasmid release specifications are critical when manufacturing many cell and gene therapy (CGT) products but current guidance defining expectations for the release of plasmids as a starting material is limited.
To reach a consensus on test attributes and release requirements for plasmids and the bacterial master cell banks used to produce them, BioPhorum members proposed a platform framework to stimulate an industry discussion. This was set out in its 2020 publication, Cell and gene therapy critical starting material: a discussion to help establish release specifications for plasmids and the bacterial master cell banks used to produce them.
Its latest paper, Further discussion on plasmids to establish release specifications using a risk-based approach to manage supply, shares industry feedback to complement CGT efforts to advance release specifications for plasmid MCBs and plasmid DNA.
A selection of feedback from the CGT community includes:
Plasmid identity testing is performed upon release from the supplier and by users before use. In the 2020 publication, Sanger sequencing and restriction digest methods were suggested. Subsequently, the team recommends using Next-Generation Sequencing techniques, if available, which have an enhanced capacity to sequence plasmid DNA and to confirm identity and cross-contamination. Alternatively, the team recommends a restriction digest assay that cleaves within the Long Terminal Repeat sequence regions (LTR) of LVV plasmid or Inverted Terminal Repeat sequence regions (ITR) of AAV plasmid to demonstrate sequence integrity.
Identity testing and contamination screening of the MCB itself is also critical. Gram stain analysis, use of selective media, and/or Analytical Profile Index (API) gallery methods may also be performed. However, 16S rRNA ribosomal sequencing of host cells provides an alternative to all three of these methods.
In-process and segregation controls are recommended to assure a low risk of cross-contamination during production.
The team proposes visual inspections to test plasmid DNA for appearance upon release and recommends that appearance should be colorless and free of particulate material. Since the plasmid is not being injected into a patient directly, considering microscopic particulates is not required and such methods should be excluded from the proposed release platform.
The general consensus among the BioPhorum team is that the percentage of supercoiling is related to transfection efficiency and productivity, but the exact relationship is unclear. The team therefore proposes a collaborative approach to collecting sufficient data to understand the correlation between supercoiling and transfection efficiency.
Since the original publication, the team has considered digital PCR (dPCR) for measuring contamination by other host cells by targeting the rRNA gene. However, there is currently no standard tool for performing the assay.
Also, there is no agreed methodology to interpret the data that dPCR assays generate. Therefore, the use of dPCR to document and record residual DNA contamination has been used cautiously within the CGT field. There is an opportunity to build industry consensus so that the dPCR assay can be used and the data interpreted in a standard way across the sector.
HPLC or SYBR GoldTM are offered as potential methods to test plasmid DNA for residual host RNA. It is generally accepted that measurement by HPLC may provide superior accuracy, precision, and sensitivity to quantify residual host RNA. However, alternative methods for quantifying residual host RNA may be advantageous, particularly for plasmid suppliers that may not have an HPLC capability.
Low bioburden rather than sterility may be acceptable in some circumstances. Unique sterility or bioburden requirements for plasmids may be defined through a risk-based approach, based on where and how the plasmids are added to the user’s processes. Either criteria (sterility or low bioburden) should be verified by the user as part of plasmid qualification and/or incoming release, based on the criticality of microbial control attributes and an assessment of existing controls.
Plasmids containing the kanamycin resistance gene and other antibiotic resistance genes are common and allow for a practical cell selection process of target cells over plasmid-free cells. Often used at working concentrations of 50–100 µg/mL in cell selection, toxicity to human health and non-resistant cell cultures necessitates some level of control and evaluation in plasmid products. Limits of detection down to 2 ng/mL in enzyme-linked immunosorbent assays may demonstrate clearance and elimination of residual kanamycin.
This selection of feedback points should stimulate a conversation and promote industry consensus.
Elemental, extractable, and leachable impurities
The number and extent of the elemental and leachable impurities associated with the plasmid and its container closure can make process development challenging. However, it may be prudent to consider the overarching critical quality attributes. In the scope of upstream manufacturing, it may be practical to assess the integrity of the plasmid material (e.g., supercoiling, percentage of nicking, identity, integrity) as an orthogonal measure of the effect of any of these impurities exerted on the material. Further exhaustive efforts in screening and characterizing these trace impurities in the plasmid material may not prove fruitful in the overall scope of manufacturing, considering the associated steps and analytical methods utilized downstream.
Significant changes for plasmids may be regarded as those that impact the quality, safety, and efficacy of the plasmids. Considerations from the ICH Q5E Comparability of biotechnological/biological products may be applied to demonstrate that changes to the plasmid manufacturing process do not adversely impact the quality, safety, and efficacy of the plasmids. The rigor around evaluating comparability should be on par with the treatment modality the plasmids are used in and in proximity to the final drug product/patient, phase of development and associated manufacturing/clinical experience and history.
Stability study design, requirements, and interpretation of results for plasmids can be acquired directly from ICH quality guidelines, including Q1A (R2) Stability testing of new drug substances and products, Q1D Bracketing and matrixing designs for stability testing of new drug substances and products, and Q1E Evaluation of stability data. While these ICH guidelines contain recommendations intended for drug substances and drug products, the concepts and suggestions can be applied to plasmids used as starting materials in CGT.
Stability testing should be done at the desired condition: –80°C or liquid nitrogen (–196°C). Testing is typically done at T0, every three to six months for the first two years, then annually. Accelerated stability testing is possible.
In the fast-moving field of CGT manufacture, there will likely be advances in applying science to plasmid release specifications in the next few years.
Following the publication of BioPhorum’s 2020 papers, the US Pharmacopoeia formed an expert panel to draft a new general chapter to standardize plasmid sourcing, manufacture, quality, and testing for plasmid DNA used in CGT product manufacturing. The EMA has also given guidance that plasmids should be made using GMP conditions.
Applying a common standard will enable communication and cooperation across the pharmaceutical industry and simplify communication with regulators.
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