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Nov 2019

Cell and Factor Therapy Lesson 4: Undefined Methodologies, Unstable Engineering and Potential Challenges in the GMP Regulatory Environment

lab technician in googles

Peter Wunderli, Ph.D., enquiry fellow, PPD Laboratories, GMP lab, discusses cell and gene therapies in an GMP environment.

The U.Southward. Food and Drug Assistants (FDA), European Medicines Agency (EMA) and other national regulatory agencies have provided guidance for the development, manufacture and evaluation of factor therapy medicinal products (GTMPs). These guidelines ensure appropriate good manufacturing do (GMP) systems are in place to satisfy the mandates for condom, identity, forcefulness/potency, purity and consistency (all product quality attributes) of pharmaceutical products. Equally more than products progress to clinical trials and agencies and industry proceeds experience in developing GTMPs, initial, updated typhoon and concluding guidance documents are being issued that reflect the evolving perspectives and expectations agencies have for evaluating these products. As a result, a very skilful overview, "Regulatory considerations for factor therapy products in the Eu, Nihon and the US"i, published in 2017, is already outdated past more recently issued typhoon and concluding guidance2-vii.

The purpose of this review is to identify some areas where regulations on specific methodologies are not defined, technologies are considered less than ideal or where contempo developments have added some potential challenges to the characterization of these products. The specific areas discussed hither are methods directed at the quality attributes associated with establishing strength/authority, purity and safety of GTMP products, and especially viral vectored products.

The first of these, strength/dominance, is of paramount importance, not only because these are required under 21CFR sections 600.3(south) and 210.iii(b)(16), but considering cistron therapy vectors are directed at therapeutic, prophylactic or diagnostic applications and function past inserting or modifying genes in target cells and tissues. If this results in likewise much expression, there could be significant risks to patient health from possible toxicities. Too footling expression, and the treatment could fail to offer the intended wellness benefit. In that location is too potential adventure both from how it is delivered and what happens to the genetic material later on it enters the cells. Further, potential host responses to the commitment might significantly impact safety, efficacy and durability of the intended disease treatment.

For typical poly peptide based biotherapeutic entities, conclusion of product potency is already circuitous, requiringin vivo orin vitro determinations of relative say-so (ideally targeting the known or theoretical mechanism of action of the drug) that allow comparison of product activeness to a reference material.  By comparison, GTMPs brand that requirement seem uncomplicated, as there are multiple indicators of product potency, including:

  1. Physical titer – representing the number of copies of the gene of interest (GOI) inside a quantity of the production;
  2. Infectious titer – establishing the concentration of those genomes than can gain entry to the jail cell and replicate;
  3. Gene expression – demonstration that the gene not but enters the prison cell, only can direct cellular expression of that genetic textile in a way that can be measured and is dose-dependent;
  4. Functional potency – the expression product of the cistron is shown to have the GOI's targeted activity.

There are also multiple ways of measuring each indicator of potency. FDA guidance8 acknowledges that "the complexity of CGT products tin present significant claiming(s) to establishing potency assays." The guidance goes on to say that meeting all the requirements may not be possible in early on phase clinical investigations and that a unmarried test cannot fairly measure the product attributes that predict clinical efficacy. However, information must be available and evidence substantial "to clinch the identity, quality, purity and strength … during all phases of clinical written report." Regulatory agencies strongly encourage development and application of expression and functional methods while products are in development to support indication of new drug (IND) applications and recommend discussions with the appropriate agency as potency measurements are designed, evaluated and validated.two,7,8,9

At a public meeting in December 2018, the FDA'south Blood, Vaccines and Other Biologics advisory commission held a workshop titled "Quantitation of AAV-Based Gene Therapy Products" to review and discuss best practices for physically quantitating the concentration of recombinant adeno-associated viral (rAAV) vectors present in a production. Standard quantitation has been based primarily on methods applying quantitative PCR (qPCR) every bit it has a long history and is in widespread use. Still, droplet digital PCR (ddPCR) and size exclusion chromatography (SEC) methods were reviewed and discussed at length. Denise Gavin, interim gene therapy branch chief, partitioning of prison cell and cistron therapy, CBER, U.South. FDA, provided the agency's perspective. She made no recommendations for any specific method, merely suggested that product specific processes should be considered and that the agency recommends early on development and optimization beginning in preclinical production evolution to ensure that the data provided demonstrate suitability and tin back up comparative dosing across production lots and clinical testing.

Because it is understood that not all vector particles that contain the GOI in a product may exist capable of inbound cells and activating the genetic fabric, transfection or transduction assays are applied to decide the specific activity of the delivery vectors every bit a ratio of the concrete number of genomes/particles (established by the methods described to a higher place) to those capable of successfully transfecting or transducing cells.  Methods can involve the use of helper virus and have evolved from titration assays with rather subjective cellular cytopathology determinations to those that found the presence of the vector delivered product (via qPCR or ddPCR). As these methods have inherent variability that may be compounded by variability of vector production and purification processes, other assays are suggested and are in development equally more than accurate or less variable alternatives to support evolution of viral vectored products.10

Regulatory agencies also acknowledge that production complexity may limit the ability to know or demonstrate the mechanism of activeness of gene therapy products. Nevertheless, some measure of "the level of transgene expression, associated biological activity, and factors associated with the proposed mechanism of action of the vector/delivery organization including maintenance of the therapeutic sequence in the target cell should be analyzed7;" and "the potency assay should incorporate both a measure of factor transfer and the biological result of the transferred gen."8 Both the USFDA and EMA take that measuring the functional action might not be possible, and propose that multiple assays might be necessary.vii,eight They suggest that "immunochemical methods to determine the integrity and quantity of an expressed protein product" can "supplement" functional activity7 and hash out "progressive potency analysis implementation" and detailed evaluation and modification of such assays every bit the product development process continues.8

Viral vector products, and rAAV in item, may exist a heterogeneous mixture of empty capsids (i.e. do not contain Deoxyribonucleic acid), uninfectious particles (i.e. incorporate DNA, but DNA amplificationin-vitro is not observed) and infectious particles (enters the cell and transgene expression/DNA amplification is observedin-vitro).9 Particles that do not upshot in expression/amplification are considered product- related impurities that can bear upon product immunogenicity and need to be quantified.two,9 Production atmospheric condition and purification processes can dramatically impact the levels of these impurities, and there is some debate around the touch on of these impurities on product functioning,eleven but the regulatory perspective of these particles as contaminants suggests attempts should exist fabricated to at least reduce, if not eliminate, non-transgene expressing particles.

Residual nucleic acid material from production cells and/or plasmids/helper virus present within or external to capsids or other delivery vehicles is too a concern. Both the size and quantity of these contaminants pose risks of either unintended transfer of a gene with functional expression capabilities or genetic textile capable of integrating with the chromosomes of recipient cells and altering cell function. Such events could upshot in agin events in patients. Guidance requires selection of jail cell lines and helper sequences to reduce risk and that product related impurities "be identified and their levels quantified"7 and that process related complexed nucleic acids "be addressed with respect to their bear upon on safety and performance of the complex when administered to the patients."2  The FDA recommends testing for such impurities, optimization of manufacturing processes "to reduce non-vector Dna contamination" and to "monitor and command the amount of extraneous nucleic acid sequences."2 While the extent of the identification of the impurities required by agencies is still evolving, there are growing indications that agencies accept an expectation to at least assess the quantity and the size distribution of non-transgene nucleic acid fragments contained inside the product.

Another condom expectation of the agencies is to assess viral vectored cistron therapies for the power to replicate within cells.2,vii The FDA has issued a specific guidance on these methods for lentiviral vectors,3 but this expectation applies to all viral vectored products and extends to products that are generated using insect cell/virus platforms. It is a requirement for drug substance lot release and, for retro- or lentiviral GTMPs, extends into follow-upwards assessment in patients. Whether second- and 3rd-generation vectors designed to forbid spurious recombination and potential vector variant replication can sufficiently demonstrate their ability to eliminate that risk and the need for such testing remains to exist seen.

Recent information regarding the presence of replication-competent insect rhabdovirus within some Sf9 cell banks (Sf-RV),12 including those used for industry of viral vectored gene therapies, was discussed at the Leap 2019 ISBioTech coming together. Robin Levis (deputy director, division of viral products from the office of vaccine research and review, CBER, FDA) publicly suggested that this should be considered by the agency and drug product manufacturers. While purification processes for standard biological products are validated for their general ability to remove potential accidental virus particles, viral vectored GTMPs obviously can't practice and then.  This does effect in an expectation that testing for residual Sf-RV nucleic acid will be performed in such products, but should product release testing be extended to include methods to detect the presence of replication competent Sf-RV?

GTMPs are generally very complex products that have already shown their potential to dramatically change existing handling modalities and provide options for rare and genetic diseases that did non previously be.  Like the evolution of regulatory expectations for what are now considered standard biotherapeutic products and the building upon lessons learned from those products, GTMP regulations will also continue to evolve. How regulations of GTMPs evolve will depend upon safe profiles established from the long-term follow-upward of already approved products, responses to issues identified from subsequent authorizations and the results of experimentation reported past developers in their efforts towards characterizing these products for potency, purity and safety. The all-time recommendation for being aware of these changes is for GTMP drug developers to start communications with regulatory agencies early in the process and to meet regularly to ensure their development plans remain aligned with agency expectations.

  1. Regulatory Considerations for Gene Therapy Products in the US, Eu and Japan. https://www.researchgate.net/publication/321951805_Regulatory_Considerations_for_Gene_Therapy_Products_in_the_US_EU_and_Japan
  2. Chemical science, Manufacturing, and Control (CMC) Information for Homo Factor Therapy Investigational New Drug Applications (INDs), Draft Guidance for Industry, FDA,  July 2018 https://world wide web.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-factor-therapy-investigational-new-drug
  3. Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Production Manufacture and Patient Follow-Up, Draft Guidance for Industry, FDA,  July 2018  https://www.fda.gov/media/113790/download
  4. Homo Gene Therapy for Rare Diseases, Draft Guidance for Industry, FDA, July 2018 https://world wide web.fda.gov/media/113807/download
  5. Homo Cistron Therapy for Retinal Disorders, Typhoon Guidance for Industry, FDA, July 2018 https://world wide web.fda.gov/media/113807/download
  6. Man Gene Therapy for Hemophilia, Draft Guidance for Industry, FDA, July 2018 https://www.fda.gov/regulatory-data/search-fda-guidance-documents/homo-gene-therapy-hemophilia
  7. Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal, EMA/True cat/80183/2014 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-quality-non-clinical-clinical-aspects-gene-therapy-medicinal-products_en.pdf
  8. Potency tests for cellular and cistron therapy products, Guidance for Industry, FDA, January 2011 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/authorization-tests-cellular-and-gene-therapy-products
  9. Reflection paper on quality, non-clinical and clinical issues related to the development of recombinant adeno-associated viral vectors, EMEA/CHMP/GTWP/587488/2007 Rev.1, June 2010 https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-newspaper-quality-not-clinical-clinical-bug-related-development-recombinant-adeno_en.pdf
  10. Accurate titration of infectious AAV particles requires measurement of biologically agile vector genomes and suitable controls, Molecular Therapy, Methods and Clinical Development, 2017 https://www.prison cell.com/molecular-therapy-family unit/methods/pdfExtended/S2329-0501(18)30067-6
  11. AAV empty capsids: for improve or for worse? Molecular Therapy, 2014 https://www.researchgate.net/publication/259566547_AAV_Empty_Capsids_For_Better_or_for_Worse
  12. Assessing and addressing the risks associated with Sf-Rhabdovirus, an accidental amanuensis in the baculovirus-insect cell system, Am Pharm Sci, 2014 http://www.americanpharmaceuticalreview.com/Featured-Articles/190742-Assessing-and-Addressing-The-Risks-Associated-With-Sf-Rhabdovirus-An-Adventitious-Amanuensis-In-The-Baculovirus-Insect-Cell-Organisation/