OR WAIT null SECS
There is growing pressure for robust and economically scalable viral-vector manufacturing technologies.
Biopharma drug developers are increasingly realizing the potential of cell and gene therapies (CGTs) using viral vectors as gene delivery vehicles. Rapid growth in cell-based immuno-oncology pipelines and early development efforts with in-vivo lentiviral vector (LVV)-based therapies are fueling CGT growth. As a result, there is increasing pressure for robust, scalable, and cost-effective good manufacturing practice (GMP) viral vector manufacturing platforms.
In exploring the tactics and strategies needed to develop a robust manufacturing platform designed to address these growing needs, new manufacturing platforms are being developed. The developed platforms aim to overcome the numerous limitations associated with traditional serum-dependent and adherent-based LVV production processes while offering scalability. The necessity of accelerating production timelines and allowing ease of transition from product development (PD) to GMP manufacturing must be kept in mind throughout the process development stage to support production of high-quality LVVs in a cost-effective and time-efficient manner.
In the past 20 years, innovations in molecular biology and genetic engineering techniques have greatly advanced CGTs. As drug developers have increasingly realized the potential of these revolutionary therapeutics, CGTs have become one of the fastest-growing areas of therapeutics in the biopharma industry. Once reserved for rare and orphan diseases, CGTs in the development pipeline now target a broad range of therapeutic areas, from various cancers and metabolic diseases to sensory issues (1). The success of these therapies is reflected in the size of the global CGT market, which is anticipated to reach $42.56 billion by 2030, growing at a compound annual growth rate of 39.42% in the 2022–2030 forecast period (2).
The majority of approved CGTs rely on viral vectors to deliver genetic material to patient cells, helping to overcome genetic abnormalities resulting in disease (1). LVVs possess characteristics that lend themselves well to this task, offering the ability to integrate genes into the genome of the target cell for long-term expression. In fact, the most common technology used in the genetically modified cell therapy pipeline currently—chimeric antigen receptor (CAR) T therapy (2)—typically relies on LVVs or retroviral vectors to introduce CAR genes to patient T cells ex vivo. Once the T cells are reintroduced to the patient, the CAR enables specific and targeted destruction of cancer cells by the immune system.
Innovative research has continued to expand the capabilities of LVVs, with the CGT development pipeline seeing the potential for in-vivo LVV therapies, which could offer a more cost-effective treatment option for a larger patient population. However, this application of LVVs requires significantly more effort to establish a process for delivering vectors with high-yield, high-quality, and in larger amounts.
There is great potential for LVV therapeutics to treat a wide array of diseases and to significantly improve the lives of patients. However, as LVV drug modalities evolve, new and possibly unfamiliar challenges can arise. Developers and manufacturers taking on the challenge of LVV production must establish processes that consider the rising demands for higher purity and stability.
Administration of in-vivo viral vector CGTs via direct infusion is typically associated with product safety profile requirements that differ from ex-vivo therapies. Following ex-vivo LVV transduction, patients’ cells will undergo further processing and cryopreservation to prepare and store the cells prior to intravenous infusion. As a result, if impurities such as residual host cell DNA, host cell proteins (HCPs), and residual plasmids are present in the LVV product following fill/finish, they are less likely to have a detrimental impact on patient safety than if directly administered.
Developers must therefore optimize downstream processes in LVV manufacturing with purity in mind, ensuring that residuals and impurities that could put patient safety at risk are absent from the final product.
Compared to other viral vectors such as adeno-associated virus (AAV), LVVs are typically more fragile, being sensitive to a variety of chemical and physical conditions, such as temperature, pH, conductivity, and centrifugal force. Outside of optimal conditions, LVV efficacy, stability, and yield can be heavily impacted. Throughout LVV production, vector loss often occurs during filtration or processes where there is a risk of high shear force. Freezer storage throughout production is not an option due to the sensitivity of LVV, meaning harvest-to-fill/finish must typically be accomplished without intermission.
LVV production processes, whether for ex-vivo or in-vivo therapies, therefore, need to be carefully developed to minimize yield loss and maintain stability.
As LVV-based therapies become more complex, developers and manufacturers must consider how production processes will ensure safety and purity, as well as the need to maintain stability. To meet the anticipated rising demand for LVV therapies, these processes will also need to offer several other key features, including scalability, increased productivity, accelerated timelines, and more cost-effective manufacturing.
Rather than relying on traditional LVV production processes based on serum-dependent and adherent culture-based platforms, meeting growing demand will likely require CGT developers and manufacturers to look to alternative options. A platform process for scalable LVV manufacturing based on transient transfection of serum-free suspension growing cells can be an effective solution to the rising demand for viral vector therapies.
Suspension cell culture
The use of mammalian cell lines such as human embryonic kidney 293 (HEK293) cells adapted to grow as suspension cell cultures generally offer greater scalability compared to production in adherent cell lines. The traditional 2D culture vessels used to grow adherent cell cultures during upstream processing (USP) in LVV manufacturing require extensive manual handling and operation. Being labor-intensive, the use of these culture vessels for adherent HEK293 cell line growth increases product cost per dose while also making scaling more challenging. Increasing batch volume will typically require “scaling-out” in an adherent process, necessitating the accumulation of vessels that can require significant amounts of space and labor hours.
On the other hand, suspension cell cultures are associated with minimal manual handling and more linear scalable processes within bioreactors. The ability to easily scale up USP with suspension enables LVV manufacturers to simplify development towards a commercial-ready process.
Traditionally, LVV production in cells requires serum-containing media—for example, media for LVV manufacturing using HEK293 cell lines will include fetal bovine albumin (FBS) to facilitate cell growth. Because this product is animal-derived, however, there are safety concerns around its use, with the potential for the introduction of prion proteins and subsequent risk of encephalopathies if not irradiated. Batch-to-batch variation can also be an issue due to FBS’ animal origin, making its composition difficult to control.
Alternatively, LVV developers should consider designing production platforms using chemically defined, serum-free media to minimize these safety and variation risks.
As well as incorporating serum-free media and suspension cell cultures into USP during LVV manufacturing, drug developers will need to consider how all processes in the platform could be optimized with scalability, productivity, safety, purity, stability, and speed in mind. A successful LVV manufacturing platform will consider not only the needs of different potential LVV products and construct designs but also the flexibility to be easily adapted for use in various CGT applications, whether in vivo or ex vivo, encoding CAR or other constructs. All of these factors must be taken into account to build a robust, scalable, and GMP-compatible manufacturing platform capable of supporting a variety of projects.
Process optimization considerations
Optimizing critical steps in USP will encompass yield maximization at harvest. For downstream processing (DSP), processes should be optimized for the removal of residuals and impurities to minimize vector loss.
Selecting production parameters and optimizing for robustness and scalability will rely on the appropriate design-of-experiment (DoE) studies. For example, DoE studies could be used to determine various upstream and downstream factors of operations. Additionally, scale-down models can be used to project upstream and downstream scaling, allowing issues raised to be identified, investigated, and solved at an early stage. Steps requiring optimization can also be quickly pinpointed.
It is key that both USP and DSP operations aim to preserve the critical quality attributes of the product. Ultimately, success will present as a product with high infectious titer (> 1E7 TU/mL), a low particle-to-infectious ratio (typically < 200 particles/TU), efficient transduction of target cells, and minimal HCP, host cell DNA, and plasmid DNA impurity content.
Further accelerating timelines
There is potential for many bottlenecks throughout LVV manufacturing that can cause delays and lengthen timelines. Product release and associated release testing for tech transfer are common causes of bottleneck situations. By having the supporting analytics to accommodate release testing requirements, as well as analysis needed throughout PD, the platform can be streamlined to prevent delays. Bottlenecks during tech transfer can be further minimized by ensuring that all equipment and consumables align with GMP standards, including single-use technologies.
As the capabilities of LVVs expand and increasingly challenging LVV-based therapeutics enter the development pipeline, manufacturers will need to continue to consider how best to support a myriad of diverse projects. Achieving this will rely on the development of a robust LVV manufacturing platform, optimized with the purity, safety, and stability of these products in mind. For these LVV-based products to attain commercial success, the platform must also offer scalability and accelerated timelines, while being cost-effective. By working with experienced strategic partners that provide end-to-end solutions, drug sponsors can be assured the unique needs of their LVV project will be met.
1. ASGCT and Citeline. Gene, Cell, & RNA Therapy Landscape: Q3 2022 Quarterly Data Report; American Society of Gene & Cell Therapy and Citeline. October 2022.
2. Vision Research Reports. Cell and Gene Therapy Market (By Therapy Type: Gene Therapy, Cell Therapy; By Application: Oncology, Genetic Disorders, Dermatology Disorders, Musculoskeletal Disorders, Others; End-Users: Hospitals, Cancer Care Centers, Wound Care Centers, Others)–Global Industry Analysis, Size, Share, Growth, Trends, Regional Outlook, and Forecast 2022-2030; Vision Research Reports. 2022.
Bojiao Yin is director; Mercedes Segura Gally is vice-president; Jimmy Xin is scientist; and James Fasano is scientist; all at Vector Process Development, ElevateBio.
Vol. 36, No. 3
When referring to this article, please cite it as Yin, B.; Gally, M.S.; Xin, J.; Fasano, J. Tactics and Strategies for Designing an Ideal Lentiviral Vector Platform. BioPharm International 2023, 36 (3), 18–21.