Demand for plasmids is rising dramatically as large numbers of messenger RNA (mRNA) vaccines and viral-vector-based therapeutics advance through clinical trials toward approval and commercialization. The upstream manufacturing process is an important part of bringing plasmid DNA (pDNA) materials to market, as achieving the highest yields possible at large scale is essential to meeting future demand. Fermentation is anticipated to be the predominant method for large-scale production of high-quality pDNA products.

Upstream plasmid production: fermentation, harvest, and lysis

For conventional biologics produced via fermentation, upstream operations have been associated with fermentation and harvesting. In the context of plasmid bioproduction, however, it is also common to include cell lysis and clarification as part of the upstream process, according to Nuria Gomez Santos, head of process and analytical development for pDNA with Catalent Cell & Gene Therapy. “The cut off is established based on the presence (upstream) and absence (downstream) of bacterial cells or cell debris in these process unit operations,” she explains. Catalent applies such a cut off using a 0.2-micrometer filtration step to further segregate upstream and downstream operations.

Some companies, says Karl Varadi, lead process development manager for pDNA and mRNA at AGC Biologics, regard resuspension of harvested biomass, subsequent alkaline lysis, clarification, and concentration/diafiltration as midstream processing (MSP), with capture, polishing, with other related purification, formulation, and fill/finish steps are part of downstream processing. Given the importance of lysis, neutralization, and clarification in pDNA production, John Bowen, senior director for Nucleic Acids and Plasmid Operations at The Center for Breakthrough Medicines, agrees that these steps should be thought of separately, as mid-stream unit operations.

For the purposes of this article, however, lysis and related activities will be included in the discussion of upstream processing for pDNA manufacturing.

R&D, GMP-like, and GMP plasmids do not impact upstream operations

The expectation that drug manufacturers take a risk-based approach to production has drawn attention to the manufacturing practices employed for key materials used in the production of drug products, even if they are not intended to be present in the final drug product. Such ancillary materials include plasmid DNA used as a template for the production of mRNA and for the production of viral vectors used for delivery of genetic cargo. In general, the industry is moving towards the use of the highest-quality materials for both clinical and commercial manufacturing of not only critical raw materials, but ancillary materials as well. Most pDNA manufacturers consequently offer three different product grades for phase-appropriate use: research, intermediate, and current good manufacturing practices (CGMP).

“The different quality grades are aimed at different applications,” states Martin Schleef, CEO of PlasmidFactory. “For initially testing a new therapeutic gene candidate or gene delivery strategy, a research-grade plasmid would be sufficient. Further refinement of the research process results in the intermediate and highest, GMP-grades for clinical and commercial applications,” he observes.

Plasmids for use in mRNA and viral-vector manufacturing may be intermediate- or GMP-grade, depending on the preference of the drug manufacturer. Plasmids used in direct clinical applications, such as for DNA vaccines, must be of GMP-grade, according to Marco Schmeer, project manager at PlasmidFactory.

“The higher quality grades,” Schleef says, “generally are produced in dedicated manufacturing spaces, undergo more extensive quality-control testing as part of an active quality management system, and have greater documentation packages.”

The quality grade does not have significant impact on upstream and downstream process operations, however, according to Santos, as the same platform process is executed to generate R&D, GMP-like, and GMP-grade plasmids. Indeed, there should be no impact on process development if the upstream process is created with manufacturing considerations in mind from the outset, Varadi agrees. “A well-defined upstream process is ideally initiated and designed via down-scaling of an existing GMP manufacturing infrastructure, in order to compensate for technical differences. This approach allows for the generation of a robust and easily scalable process, which then just needs completion by GMP/GMP-like documentation,” he comments.

At Catalent, for instance, R&D-grade material is generated using a downscale model operated with the same parameter set points as in its cleanrooms, Santos notes. This representative R&D material has been used for process development purposes and pre-clinical studies. Both GMP-like and GMP-grade plasmids are produced using fully traced raw materials and fully disposable qualified equipment. Operations are identical as only the shared documentation and level of quality oversight varies between these grades.

From process optimization to bacterial strain engineering

The increasing amount of pDNA required for therapeutic and vaccine applications has drawn plasmid design and fermentation strategies into the spotlight. To help prepare for the expected significant market demand, the increasing amount of required pDNA for therapeutic and vaccine applications has drawn plasmid design and fermentation strategy into the spotlight, Varadi contends.

Plasmids are typically produced recombinantly in E. coli strains through a high biomass fed-batch fermentation process. High titers can be achieved through multiple means by deregulating the repression on plasmid replication, according to Matthias Craig, senior manager for Product Development at Catalent Cell & Gene Therapy. “High yielding plasmid fermentation processes have been developed and fine-tuned over the past two decades,” he states.

Implementation of fed-batch protocols has led to improved pDNA yields and increased batch success rates and optimized cost efficiency, Varadi agrees. He adds that understanding the molecular mechanisms that modulate the plasmid copy number and implementing them in next-generation plasmid backbones will help further boost yields for plasmids with more problematic genetic payloads.

“Fermentation has indeed become more sophisticated to achieve the desired goals in plasmid production such as plasmid yield per biomass,” Schleef observes. He cautions, though, that more complex issues like maintaining plasmid structural stability still remain to be addressed.

Some of the biggest factors for upstream pDNA process development include media optimization, fed-batch strategy, and Escherichia coli (E. coli) strain selection, Bowen comments. Engineering of improved E. coli host strains has, according to Craig, to some extent become more of a focus recently than fermentation process optimization. “These strains are characterized by higher fermentation titers and/or lead to increased stability of nucleic acid sequences that are prone to deletion/recombination events, such as inverted terminal repeats (ITRs) and long polyA tails,” he explains.

Another important advance noted by Craig is the development of both batch and continuous cell lysis approaches that afford high recoveries and accommodate a wide range of production scales. The adoption of single-use equipment in upstream operations has, meanwhile, significantly reduced the probability of cross-contamination, which is very important in a multiproduct facility producing a multitude of plasmids, Bowen observes.

There are many challenges that manufacturers must navigate when implementing upstream processes for pDNA production. First and foremost, very high volumetric yields are achievable for pDNA, but such yields are much lower than those achievable for overexpressed, non-toxic recombinant proteins, says Ram Shankar, manager of R&D for PlasmidFactory.

“That being said, simply overproduction of plasmid per cell would not go very far if plasmid stability and homogeneity cannot be guaranteed. Such aspects have to be considered when designing an upstream process for plasmid production,” Shankar notes. “In this context,” he continues, “we have developed strategies that keep productivity high without having to rely on antibiotics while controlling for the maintenance of critical sequence structures in the product (e.g., ITR sequences for productive adeno-associated virus [AAV] manufacturing or polyA tracks for mRNA manufacturing).”

Another common issue, according to Varadi, is achieving high-yield manufacturing of plasmids that contain difficult genetic payloads (gene-of-interest size, base composition, toxicity, use of selection markers). Larger plasmids that impose a significant metabolic stress on the host cell, meanwhile, can reduce culture fitness, leading to bad growth and low plasmid copy number. He adds, though, that narrow control of carbon and nitrogen source feeding and use of alternative non-antibiotic selection markers can reduce metabolic burden on the host cell.

It is recommended, Craig emphasizes, that scientists and drug developers verify that their plasmid constructs are of very-high copy number nature. “This characteristic is associated with the presence of a single-point mutation in the plasmids’ origins of replication and further derepresses plasmid replication, leading to up to a five-fold increase in plasmid titers,” he explains.

Leaky promoters that lead to pre-mature protein expression during the growth phase can also be detrimental to upstream plasmid success, Varadi comments. He adds that some large pDNA losses can also occur during alkaline lysis. In fact, a well-defined alkaline lysis step is far more important in reducing plasmid loss than avoiding cyclic performance during downstream chromatography operations, he contends.“The lysis step has several critical parameters that need to be well balanced, like buffer volumes, buffer contact time time, and applied shear forces during mixing,” Varadi explains.

Efficiency and productivity of upstream plasmid processes

Plasmid DNA fermentation and cell lysis are complex processes that can be influenced by many different process parameters. Both plasmid titer and quality, including supercoiled plasmid content, can be directly affected, according to Santos. “Proper control of plasmid replication repression and induction is key to reach high volumetric and specific yields. In the fermentation process, this is achieved through utilization of appropriate culture media, robust control of the bacteria growth rate, appropriate temperature set points, and the availability of oxygen,” she says. For cell lysis, Santos stresses that it is crucial to recover as much material from the E. coli biomass as possible without degrading the product. “A fine and well-balanced alkaline lysis approach is the preferred unit operation to meet this objective,” she observes.

There are several steps that can be taken to boost efficiency and productivity of pDNA fermentation and lysis processes. During cell-line development, reducing cell stress in general improves the quality and quantity of pDNA produced, according to Varadi. During fermentation, reduction of metabolic burden and the use of well-designed fed-batch profiles is highly beneficial, as lowering metabolic burdens leads to higher product yields.

Closer real-time monitoring of critical parameters during batches, such as online cell density measurements, and comparison of multiple production runs both play an important role in improving plasmid fermentation processes, according to Shankar. “Implementation of process analytical technology tools for various critical process (off-gassing) parameters in combination with at-line analysis of product parameters (plasmid content and homogeneity) helps to unravel hidden impacts of process variables,” he says.

One thing Varadi would like to see is the introduction by device suppliers of more scale-down (e.g., lab-scale) versions of their equipment. “Performing process development in an optimally scaled-down process makes the upstream process more reliable and robust when scaling up,” he contends.

Shift to platform plasmid fermentation processes

The successful development of platform manufacturing processes for recombinant proteins and monoclonal antibodies has created the desire to establish similar platform solutions for other biologic materials, including pDNA.

“The aim is to find one process that would fit all, rather than optimizing each process for different plasmids. This would offer cost and timeline advantages, ultimately accelerating the delivery of treatments to patients,” Craig remarks. “Operating a robust plasmid production platform that facilitates the transition of programs from a pre-IND [investigational new drug application] stage towards the clinic and to market can lead to the delivery of the required product quantities with the expected purity profiles, thereby greatly accelerating development timelines,” he adds.

That is not so easily done, however. “Development of platform processes are to a certain extent possible but there cannot be a magic bullet for every type of plasmid,” states Schmeer. “Individual plasmid size and sequence can have a huge impact on upstream process efficiency, which is often not fully appreciated,” he adds.

Output variability from platform processes, Santos agrees, is mostly associated with the specificities of the plasmid sequences themselves. “Large constructs tend to be lower yielding, and plasmids carrying repeated sequences such as ITRs and polyA tails have been documented to be more prone to deletion and recombination events,” she notes.

The solution, according to Bowen, is to develop a “flexible” platform by investigating a variety of plasmid types (size, complexity, etc.) and 

strains during process development to determine their impact on plasmid yield and quality. This will allow for flexibility when manufacturing subsequent plasmids.

Adding optimization flexibility to a robust platform process facilitates the delivery of plasmid quantities, including meeting quality specifications for challenging plasmids, Craig agrees. He also points out that timelines can be further accelerated by using off-the-shelf plasmids. “Having a well-characterized set of plasmids in advance, such as pHelper and pREPCAP plasmids, will save valuable time on subsequent applications, particularly when used in combination with a platform process,” he states.

For Varadi, developing a platform process for upstream plasmid manufacturing is not only possible, but mandatory. “A well-designed platform embraces many plasmid types and enlarges the spectrum of potential customers while reducing manufacturing costs,” he comments. One key to success, Varadi adds, is size optimization of plasmids by reducing “junk” DNA stretches within the backbone and the gene-of-interest cassette. He also notes that a combination of host-cell and plasmid-backbone mutations (already characterized in literature) is beneficial for copy-number increases that should be evaluated for industrial purposes.

Turning the tide for future upstream requirements

There is no doubt that the demand for plasmid DNA will continue to increase in the coming years. All therapy types that require pDNA as a raw material are expanding massively and require an industry in the background producing them, Varadi contends.

“Over the next few years, high-capacity manufacturing will not be the only prerequisite for pDNA manufacturers; they will also need to have the ability to produce a multitude of different constructs in smaller quantities,” Varadi observes. “High-capacity manufacturing will be necessary to meet demand, but that cannot be achieved only through upstream process optimization. Wide implementation of single-use technology and simplified scalability solutions as a link from non-GMP process development laboratories to GMP suites will be necessary,” he insists.

Scale and quality will indeed be the two key aspects that must be addressed for future pDNA manufacturing, according to Shankar. He agrees that “GMP production of plasmids through the adoption of single-use technologies for upstream processing will turn the tide of manufacturing for novel advanced therapy medicinal products.”

Cynthia A. Challener, PhD, is contributing editor to 


Vol. 36, No. 3
March 2023
Pages: 10–13, 36

When referring to this article, please cite it as Challener, C.A. Achieving High Yields in Upstream Processing of Plasmids

Articles

Cover Story

Process and plasmid design optimization, disposable equipment, and flexible platform processes all play important roles.

Achieving High Yields in Upstream Processing of Plasmids

Therapeutic biologics are thriving, according to Zhou Jiang, director Cell Culture Services at Cytiva. “As we work to advance the next generation of therapeutics, we must address these challenges to deliver the medicines patients need. The increasing diversity introduces new challenges and mandates new solutions in upstream cell culture,” says Jiang.

Gary McAuslan, CEO aCGT Vector, outlines some central challenges, stating, “cell therapy manufacturing today is unsophisticated and in the main, extremely manual and people dependent, shocking from a microbiological contamination potential, and impossible to scale in a controlled manner from a mass uptake of precision medicines perspective. Batch failures, low viability, and time to patient exceeding the patient’s life expectancy are the stark realities of today’s pharmaceutical manufacturing response that attempts to bulk manufacture what are truly individual modern medicines at a scale of N=1."

“Only one of the CAR-T innovators ship fresh patient T-Cells for good reason,” he says. McAuslan concludes. The current default, for example, for the first CAR [chimeric antigen receptors]-T Kymriah from Novartis is to use frozen T-cells that are collected at the hospital from a patient, sent across the world frozen, thawed, cell engineered, frozen again, shipped again and thawed again, before being administered inside the same hospital over a month later, taking just 11 minutes to infuse into the same patient. The current logistical complexity is restricting cancer patient access to the just 15,000 treated since the first approval in 2017 versus the projected annual 2 million patients who might benefit from CAR-T treatment. Pharma can absolutely do better than this. Both of these therapies are manufactured and shipped back frozen, a number of weeks to an individual patient for administration inside a hospital center of excellence approved to do so. Being able to eliminate these complex logistics, and time especially (in the region of 27% of patients die waiting on their own CAR-T therapy), as a complementary manufacturing supply chain will increase access substantially.”

This need for speed comes in parallel, but case by case, when compared with the warp speed efforts during COVID-19 vaccine development and deployment programs. Jiang reflects on this phase, saying, “There is also the drive to produce the highest possible titers with the best product quality, which has led to efforts to produce more robust cell lines and media formulations suitable for a wide variety of cell lines and biopharmaceutical molecules. Insights from the cell biology of CHO [Chinese hamster ovary] cells, and understanding of the relationships between specific metabolites and cell culture outputs such as titer and quality are highly valued and sought after for the realization of these goals.” This is why so much research, investment, and time has been focused on advanced therapy medicinal products (ATMPs) in the past few years.

From a drug-development and clinical perspective, analytical characterization of material is important at every step, and making sure the critical quality attributes (CQA) stay within a threshold, within specifications and guidance, are chemistry, manufacturing, and controls (CMC) issues, which need to be tackled during the early manufacturing process development. Vibha Jawa, head of Biotherapeutics Bioanalysis in Nonclinical Disposition and Bioanalysis (NDB) organization, at Bristol Myers Squibb, points to this saying, “further, we are always quantitating transduction efficiency and target response ... titer quantitation helps us to visualize the transgene expressions in a much more reliable and specific manner whether we are looking to quantitate what that transgene expression is, or whether we are looking to characterize and optimize the amount of the vector we are going to administer. This is because a lot [of] safety and toxicity challenges turn out to be proportional to the amount of the virus used, and the content of that virus from an empty versus full capsids perspective. The best case option for the patient is to have the highest does load presented via the least virus delivery method possible.” Sometimes poor results or failure here even precludes proceeding with the originally intended ATMP plan. Sometimes caregivers revert to antibody drug conjugates or other well-understood treatments, as the novel medicines are sometimes simply not the best choice option for that patient.

The closer one gets to the situation in the clinic, the more these challenges stand out in stark relief. Jawa relates that groups have been struggling to get sufficient well-characterized material for clinical studies. “Even if you have plans to file an IND [investigational new drug application], unless you have a good contract development and manufacturing organization (CDMO) ready with that kind of capacity waiting for you, you have to be able to synchronize with their timelines, because they are busy making someone else’s viral vector. So you have to put yourself into that que,” she states, referencing the need to always be cognizant of many other people’s timelines. Jawa goes on to say, “because there are very few of them [CDMOs] that are good and reliable. And you really need material that is the least toxic in terms of residual or process-derived contaminants, but is effective to get your transgene in, and is a good drug product within that viral vector.”

Another logistics and business challenge, according to Jawa, is which patient cohort one is working with. “It’s a highly competitive situation, especially if you are targeting diseases which affect large numbers of patients. For rare disease states, there is no such resource pressure, and the landscape is not as [potentially] crowded with competitors ahead of you making these vectors.”

Near patient manufacturing small footprint systems combined with increased automation seem to be where much of the current progress has been made. Baley Reeves, interim director at the National Center for Therapeutics Manufacturing, points out that, especially for small footprint systems, “with scaled-down/scale-out operations, quality control and regulatory [issues] are more difficult hurdles to manage. Automation and inline analytics become even more critical pieces of the process. This mainly impacts autologous cell therapies at the moment but could have future implications as personalized medicine takes off,” she suggests.

Common issues to achieving high titer levels are manufacturing plasmids that contain difficult genetic payloads (e.g., gene-of-interest size, base composition, toxicity, use of selection markers). When pursuing large plasmid sizes to navigate those challenges, one is then often confronted by leaky promoters that lead to pre-mature protein expression upstream, losses during alkaline lysis or repeated sequences such as inverted terminal repeats (ITR) and polyA tails that are more prone to deletion and recombination events. Some successful ways to navigate these obstacles are to optimize and balance buffer volumes, be more precise in terms of buffer contact times. By using more gentle mixing methods, one can reduce sheer impacts. And of course, it always helps to start with well-characterized pHelper and pREPCAP plasmids. Viral vector synthesis network Helper plasmid (pHelper) activates expression of the rep/cap gene on the packaging plasmid (pPackaging) and synthesis (k Cap_syn and k Rep_syn ) of viral protein (VP) and Rep protein (RepProtein). “Capsid proteins are assembled (k assembly) into empty capsids in the nucleus (EmptyCapNuc), and each capsid particle consists of 60 protein subunits. With helper functions from the helper plasmid, the Rep protein replicates (k DNA_rep) viral DNA (vDNA) from the transgene vector
plasmid (pVector)” (1).

A key to knowledge transfer may depend on reimbursement model modifications, and regulators embracing the idea of “equivalent centers,” leading to standardizing some equipment and protocols. aCGT Vector, for example, has concentrated on “right-size pharma—standardized, automated, and closed-system technology and expertise ... truly end-to-end services offering, that can increase access for innovators and patients alike. For example, Janssen with their CARTITUDE front-line defense for the most common blood cancer, multiple myeloma. Novartis also recently secured reimbursement by the National Health Service (NHS) in the United Kingdom for earlier treatment of their cancer treatment. In time as these precise targeted but individual treatments are demonstrated as a better use of healthcare budgets, the predictions point to locally distributed centers complementing large centralized facilities, where a patient can walk into their hospital, receive CAR-T treatment, and go home days later, with the hope of potentially surviving their own cancer. Excitingly, the rare disease landscape, where many of the 7000 untreatable diseases such as blood cancer, is changing and bringing hope to patients and their families,” says McAuslan.

Nguyen, T. et al. Mechanistic Model for Production of Recombinant Adeno-associated Virus via Triple Transfection of HEK293 Cells. 


Vol. 36, No. 3
March 2023
Pages: 14–15

When referring to this article, please cite it as C. Spivey. High-titer Vector Producing Cells. 

Development

Decreasing vein to vein time saves lives.

High-titer Vector Producing Cells

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 

 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 

. 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 

 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.

 viral vector CGTs via direct infusion is typically associated with product safety profile requirements that differ from 

 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.

 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.

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 

, 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.

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.

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.

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

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
March 2023
Pages: 18–21

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. 

Manufacturing

There is growing pressure for robust and economically scalable viral-vector manufacturing technologies.

Tactics and Strategies for Designing an Ideal Lentiviral Vector Platform

The pre-filled syringe industry is rapidly expanding, with a projected compound annual growth rate (CAGR) of 13.9% between 2021 and 2028 (1). Fill/finish is an intricate process requiring many steps in its procedure, from filling and stoppering to labeling and secondary packaging. Often, this intricate process is automated and includes various inspection methods. Commonly, container closure integrity (CCI) testing is left out of the automated process and is either conducted manually with outdated, probabilistic methods or not conducted at all, increasing risk of error.

In any manufacturing process, there is risk. Equipment issues and human error are unavoidable at times. In pharmaceutical production, this error equates to potential safety issues for the patient. Quantitative and deterministic CCI test methods aim to find these errors before reaching the patient, and further mitigate risks to patient safety.

Breaches in sterility come with many different defect profiles. Commonly, crack defects appear near the neck or heel of vials from impact or excessive crimping. Prefilled syringe defects form where the needle is fused to the glass or cracks in the barrel. Sterility breaches occur in the stopper/plunger area when molded poorly, or debris, such as a fiber, is present. These defects may allow bacteria and microbial ingress into the product, voiding sterility and increasing patient risk.

Biologics are typically high risk, costly, and created in small batches. Along with being parenteral-based products, biologics’ sensitive nature requires CCI to avoid recalls, product loss, and life-threatening patient risk.

Proper CCI testing of biologics must be non-destructive, quantitative, deterministic, scalable, and capable of detecting defects per manufacturer risk assessment. Adhering to these requirements leaves few CCI testing methods. Vacuum decay and MicroCurrent high voltage leak detection (HVLD) are not only the most popular CCI methods, but two of the most effective scalable technologies.

Best utilized when testing small-molecule products, vacuum decay has cemented itself as a go-to method for CCI. Vacuum decay as a CCI testing method is recognized by ASTM F2338, USP 1207, and is an FDA consensus standard (2). Utilizing a vacuum source, a vacuum is applied to a test chamber. Vacuum is then held within the test chamber for a period of time during the equalization stage, and then moves into a test stage where the loss of vacuum is measured by a transducer. Vacuum decay is not only suitable for lyophilized products with large amounts of headspace, but beneficial for liquid leaks as well. The difference in headspace versus liquid leaks is the amount of vacuum required to conduct the test, as well as the physical attributes of the product inside the package system. Headspace leaks are typically tested at 500mbar of vacuum. This allows measurement of the gas inside the package without evacuating the entirety of the volume during the evacuation stage. Liquid leak detection is conducted at high vacuum, less than 5mbar, where the goal is to vaporize liquid and measure the change in gas pressure.

Although suitable with a large quantity of applications and product types, vacuum decay struggles to detect microleaks in packages containing large-molecule solutions. The issues with vacuum decay are not left unsolved with HVLD standing at the forefront of CCI methods for large-molecule products.

MicroCurrent HVLD utilizes a high voltage and detection probe to scan the glass or polymer parenteral for potential breaches in sterility. The parenteral acting as a capacitor is rapidly spun, allowing the liquid product to coat the inside walls of the parenteral. Defects in the parenteral cause a large spike in voltage as liquid is detected. Unlike traditional HVLD, MicroCurrent HVLD utilizes less than 50% voltage and produces nearly no ozone. These innovations combined with extremely low current electricity prevent the breakdown of biologic products. Given that defects are detected through a liquid path, large-molecule and viscous products are able to be tested.

Both previously mentioned test methods can be used as benchtop technologies or can be fully automated into production lines. Although scalable, deterministic, quantitative, and non-destructive, they each have their limitations. MicroCurrent HVLD requires the parenteral to be cylindrical, glass or plastic, and have a minimum of a 30% liquid fill. Vacuum decay is typically only suitable for small-molecule products and may have longer testing times than HVLD. Both of these technologies are capable of testing a variety of products despite their limitations which accents their scalable nature.

These scalable technologies can be semi- or fully-automated for statistical analysis or 100% inspection of pre-filled syringes or other parenterals. When conducting CCI testing, scalability is a necessity. In-depth analysis and laboratory-based testing should match inline testing. Using the same validation ensures automated systems are working in a robust, repeatable, and reliable manner. Scalability can be broken down into three main
categories: laboratory, semi-automated/automated statistical sampling, and 100% inline testing. The difference between these three methods of scalability is the frequency of testing. High-testing frequency ultimately reduces risk of not only recall, but potential patient harm.

The European Union’s Annex 1’s 2022 revision aims to increase testing frequency for products with high defect rates (3). The revision specifies all fusion sealed, small-volume parenterals must be subjected to 100% CCI testing with a deterministic method. The regulatory agency notes that visual inspection is not an approved deterministic inspection method. This new guidance has driven the need for automated and semi-automated inspection during times of high labor costs and labor shortages.

Automated systems host a multitude of benefits, but greatest of all is their ability to increase testing frequency and limit human interaction. Limiting human interaction assists in mitigating human error. As the technology advances, risk decreases, providing a higher quality product and a safer pharmaceutical industry. The industry cannot exceed its current state without keeping the science of quality in mind.

Prefilled Syringes Market–Global Industry Analysis, Size, Share, Growth, Trends, and Forecast, 2021-2028

ASTM. Standard Test Method for Nondestructive Detection of Leaks in Packages by Vacuum Decay Method. Dec. 2, 2020.

EU GMP Annex 1 : Manufacture of Sterile Medicinal Products

When referring to this article, please cite it as T. Harris. Automated Inspection of Pre-filled Syringes and Biologics During Fill/Finish. 

Shifts in pharmaceutical packaging have spurred tremendous growth in the pre-filled syringe fill/finish industry. 

Automated Inspection of Pre-filled Syringes and Biologics During Fill/Finish

For several years after Crick (1) first described the genetic role of RNA, this molecular entity was perceived simply as an “inert” carrier between DNA and protein. Today, however, this view has almost entirely changed, and RNA-based molecules have been implicated in a broad range of functions including the activation/deactivation of genes, the excision of genetic material, and the transport of intercellular components. Indeed, it is expected that in the coming years, further discoveries will uncover even greater biochemical significance to this molecular type.

Given the ubiquity and variety of roles associated with RNA, it was inevitable that it would become a focus for investigators involved in the development of therapeutics. As early as 1978, Zamecnik (2) described the therapeutic use of an RNA-based oligonucleotide to inhibit replication of the Rous sarcoma virus, and, today, there are approximately 16 FDA-approved RNA therapies, 28 in clinical development, and many more expected in the near future (3).

Currently, RNA-based medicines can be segregated by their functionality and structure and include species, such as messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), and microRNA (miRNA). Other types of RNA include aptamers which are single-stranded and form higher-order structures, and more recently described, circular RNA (circRNA or oRNA), which appears to have multiple functions prior to and following the transcription process (4). Additionally, mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) constitute components of the recently developed CRISPR technology.

While not unique, the use of RNA in therapeutic applications is generally incumbered by two key issues: stability and delivery. Over many years, considerable efforts have been directed at attempts to resolve both these issues, leading to some creative solutions which, in turn, has necessitated the application of a broad range of analytics. However, one technique, namely mass spectrometry (MS), is particularly notable for: its ability to provide analysis of all RNA types, regardless of size and modification; the fact that it can be used for qualitative as well as quantitate investigations; the technology’s ability to be applied in both research and quality control (QC); and its applicability in the area of delivery systems.

Since the late 1970s, when both therapeutic biopolymers and analytical MS began a decades-long connection, there have been some considerable advancements in both therapeutic development and analytics. Most early biopolymer MS was applied to peptides and proteins, and, today, this technique has become arguably the single most important analytical tool in this field. Indeed, several laboratories within SGS, for example, were founded by one of the early innovators, H.R. Morris, whose contributions included the first combination of high-field magnet technology with soft ionization (5) and some of the first protein-mapping studies (6). These laboratories and others continue to encourage the adoption of such technological innovations, which have resulted in the application of advanced analytical services in support of the most challenging biopolymer studies.

Many of the advances made in protein MS have been adapted to make the technique appliable to the study of oligonucleotides, where attributes such as sequence, modification, and quantitation are critical.

’s report in 1992 (7), there has been increased attention given to the application of MS-based technologies in the analysis of RNA, including top-down methods that provide data from intact RNA species. In 2012, Taucher and Breuker (8) were amongst the first to report sequence coverage of full-length transfer RNA (tRNA) using Fourier transform ion cycloctron resonance (FT-ICR), combining data from electron detachment dissociation (EDD) and collision-activated dissociation (CAD) experiments. However, while such MS-based studies continue to be explored, they remain constrained by several somewhat related factors, such as the large molecular size, the inability to distinguish different species with the same mass, the high degree of purity required, and the limited availability of software to support top-down RNA analysis.

Perhaps to a lesser extent, these limitations also apply to top-down protein studies, although in this field these constraints have largely been overcome by mapping techniques. This approach involves controlled and specific digestion of the protein to yield a peptide mixture that can then be analyzed by a variety of MS-related methodologies, the most familiar being liquid chromatography–mass spectrometry (LC–MS). It was inevitable that such an approach would evolve in the RNA field, and this has now been successfully applied to species such as the mRNA coding for the SARS-Cov-2 spike protein (9). Such methods rely on digestion (partial and complete) with RNAses such as T1, RNAse A, and MazF prior to analysis of the products by LC–MS/MS. This is a very effective strategy because it can be automated with the use of immobilized enzymes (9), provides sequence confirmation as well as the ability to conduct de novo investigations, is able to identify and locate post-transcriptional and process modifications, can be used qualitatively and quantitatively, and may be applied to bioanalytical applications.

In contrast to polymerase chain reaction (PCR) methodologies, MS has the ability to detect and locate the more than 150 post-transcriptional RNA modifications that have been described to date. Importantly, MS is also able to identify the presence of unexpected nucleotide alterations. For many years, mass spectroscopists have taken advantage of the method’s ability to detect stable isotopic forms of elements such as hydrogen, nitrogen, carbon, oxygen, and sulfur. Nucleic acid isotope labeling combined with MS (NAIL–MS) has been used in a variety of applications to study the dynamics of RNA modification (10).

Some reports have addressed the use of MS for the investigation of dimer formation and higher-order structures, but here great care should be taken in data interpretation and extrapolation given the tendency for non-covalent molecular association within the mass spectrometer and the fundamental differences between the solution-phase environment and the environment within the mass spectrometric process. Ion-mobility MS has been used to investigate the gas-phase structures of nucleic acids such as duplexes, triplexes, and quadruplexes to try to determine if these structures are indeed similar to the structures in solution (11). Today, the use of MS for the analysis of RNA-based therapeutics has become widespread due largely to the technique’s agnostic ability to manage a wide diversity of molecular structures. From the development of Macugen (pegaptanib sodium) in the early 2000s, laboratories such as SGS and others have been highly active in analytical support for nucleic acid-based therapeutics, including today’s complex, multi-faceted structures.

Issues of stability, delivery, and binding have resulted in the development of RNA therapeutics that often involve significant modification(s) to the basic oligoribonucleic acid structure.

 provides several examples of these modified RNA structures. Changes to the phosphodiester linkage have included replacement of the free oxygen with sulfur (producing the more stable phosphorothioate linkage) and substitution with amide and boronophosphate linkages (12). Similarly, multiple, diverse structural alterations to both sugar and base moieties have been described, including methylation, methoxyethylation, fluorination, and cyclization of the 2’ sugar hydroxyl, resulting in so called “locked nucleic acid” (LNA) (10). Often, several of these modifications may be incorporated within the same therapeutic. In addition, linking of these RNA-based therapeutics with a wide range of conjugates has been exploited as a means to significantly enhance and direct their delivery and greatly improve their effectiveness. A notable example of such modification is the conjugation of siRNAs with N-acetylgalactosamine providing specific binding to asialolycoprotien receptors in hepatocytes. This structural concept has led to the successful development of the drug, givosiran, which treats hepatic porphyria by silencing the expression of aminolevulinate synthase 1 mRNA in the liver (13). Many other conjugates have been described such as lipids, peptides, antibodies (complete or fragments), vitamins, and a range of other molecular classes (14). 

Currently, the most widely used hyphenated analytical technique is LC–MS. Multiple separation methodologies have been reported including ion-pair reversed-phase chromatography, hydrophilic interaction liquid chromatography, ion-exchange chromatography, and size exclusion chromatography. A report by Santos and Brodbelt (15) provides a comprehensive review of these and other related techniques as applied to nucleic acids. Two-dimensional LC has also been described as a means to overcome incompatibility between mobile-phase composition and MS detection (16), while 3D-LC–MS—incorporating in-line digestion and hydrophilic interaction liquid chromatography (HILIC) separations—has been used for the sequencing of CRISPR-guide RNAs (17).

The majority of mass spectrometric applications involving nucleic acids have utilized either laser desorption (matrix or surface assisted) or electrospray ionization (ESI) techniques. Both are considered “soft,” thereby reducing in-source fragmentation of these large biomolecules and providing a relatively high ion current for the intact molecular species. Almost all analyzer types have been employed, including magnetic sector, time of flight, quadrupole, ion trap, and combinations of each. Today, a technology that has gained considerable acceptance both for quantitative and qualitative investigations is the so-called Orbitrap that was introduced commercially in 2005 and is shown in 

. Combining ion trap and/or quadrupole mass filters, orbitrap-based mass spectrometers provide high sensitivity with high resolution, the latter feature rising in importance as the complexity and heterogeneity of materials under examination become more acute. The implementation of multiple fragmentation functions (e.g., collision-induced dissociation, higher energy collisional dissociation, electron-transfer dissociation, ultraviolet photodissociation) on the orbitrap-based mass spectrometers also enables researchers to tackle a wide range of challenging applications. 

The use of MS in the study of oligonucleotides has become almost as significant as applications in the peptide/protein area despite the historical impact of techniques such as PCR. It is highly likely that novel RNA structures are yet to be discovered, leading to greater understanding of the function and role of this molecular type and potentially the development of more effective

therapeutic agents. As with protein chemistry, higher-order structure governs the function of many RNA species. It is likely that MS will play an important role in defining these large RNA structures that currently remain unresolved (18). Mass spectrometric techniques are just beginning to provide insights in some critical therapeutic areas where, for example, the quantitative identification of RNA modifications (genetic and epigenic) appears to provide a source of potential biomarkers and treatment monitoring in areas such as oncology (19). MS has also become important in the development and understanding of therapeutic products themselves as illustrated by the work of Packer 

 (20), who used LC–MS/MS to study mRNA modifications as a result of reaction with certain lipids used in lipid nanoparticle-vector vaccines.

There is little doubt that the past decade has seen significant advances in the understanding of ribonucleic acid functionality leading to the development of some novel and highly effective therapeutics. The growing need for sophisticated analytics to support both the investigative and routine analytical requirements of these complex therapeutics is, in large part, being met by the progression of mass spectrometric technologies. It is certain that MS will continue to maintain this close “marriage” with RNA becuase many aspects of this nucleic acid’s biochemistry are yet to be uncovered.

, 12, 138–163.
2. Zamecnik, P.C.; Stephenson, M.L. Inhibition of Rous Sarcoma Virus Replication and Cell Transformation by a Specific Oligodeoxynucleotide. 

, 75, 280–284.
3. Zogg, H.; Singh, R.; Ro, S. Current Advances in RNA Therapeutics for Human Diseases. 

, 23 (5), 2736. DOI: 10.3390/ijms23052736
4. Liu, K.S.; Pan, F.; Mao, X.D.; Liu, C.; Chen, Y.J. Biological Functions of Circular RNAs and Their Roles in Occurrence of Reproduction and Gynecological Diseases. 

, 11 (1),1–15.
5. Morris, H.R.; Dell, A.; McDowell, R.A. Extended Performance Using a High-Field Magnet Mass-Spectrometer. 

, 8, 463–473.
6. Morris, H.R.; Panico, M.; Taylor, G.W. Mapping of Recombinant DNA Protein Products. Biochem. 

, 117, 299–305.
7. McLuckey, S.A.; Van Berkel, G.J.; Glish, G.L. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. 

, 3 (1), 60–70.
8. Taucher M.; Breuker, K. Characterization of Modified RNA by Top-Down Mass Spectrometry. 

, 51 (45), 11289–11292.
9. Vanhinsbergh C.J.; Criscuolo, A.; Sutton, J.N.; et al. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. 

, 94 (20),7339–7349. DOI: 10.1021/acs.analchem.2c00765
10. Heiss, M.; Hagelskamp, F.; Marchand, V.; Motorin, Y.; Kellner, S. Cell Culture NAIL-MS Allows Insight into Human tRNA and rRNA Modification Dynamics 

, 12 (1), 389. DOI: 10.1038/s41467-020-20576-4
11. Khristenko, N.; Amato, J.; Livet, S.; et al. Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process. 

, 30, 1069–1081.
12. Halloy, F.; Biscans, A.; Bujold, K.E.; et al. Innovative Developments and Emerging Technologies in RNA Therapeutics. 

, 19 (1), 313–332. DOI: 10.1080/15476286.2022.2027150
13, Honor, A.; Rudnick, S.R.; Bonkovsky, H.L. Givosiran to Treat Acute Porphyria. 

, 57 (1), 47–59. DOI: 10.1358/dot.2021.57.1.3230207
14. Hawner, M.; Ducho, C. Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules. 

, 25 (24), 5963. DOI: 10.3390/molecules25245963
15. Santos, I.C.; Brodbelt, J.S. Recent Developments in the Characterization of Nucleic Acids by Liquid Chromatography, Capillary Electrophoresis, Ion Mobility, and Mass Spectrometry (2010-2020). 

, 44 (1), 340–372. DOI: 10.1002/jssc.202000833
16. Koshel, B.; Birdsall, R.; Chen W. Two-Dimensional Liquid Chromatography Coupled to Mass Spectrometry for Impurity Analysis of Dye-Conjugated Oligonucleotides. 

J Chromatogr B Analyt Technol Biomed Life Sci.

 Jan 15, 1137:121906. DOI: 10.1016/j.jchromb.2019.121906
17, Goyon, A.; Scott, B.; Kurita, K.; et al. On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System. 

, 94 (2),1169–1177. DOI: 10.1021/acs.analchem.1c04350
18. Amalric, A.; Bastide, A.; Attina, A.;et al. Quantifying RNA Modifications by Mass Spectrometry: A Novel Source of Biomarkers in Oncology. 

, 59 (1), 1–18. DOI: 10.1080/10408363.2021.1958743
19. Christy, T.W.; Giannetti, C.A.; Houlihan, G.; et al. Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP. 

, 60 (25),1971–1982. DOI: 10.1021/acs.biochem.1c00270
20. Packer, M.; Gyawali, D.; Yerabolu, R.; et al. A Novel Mechanism for the Loss of mRNA Activity in Lipid Nanoparticle Delivery Systems. 

Mark Rogers is global scientific director at SGS.


Vol. 36, No. 3
March 2023
Pages: 24–28

When referring to this article, please cite it as Rogers, M. The Marriage of RNA and Mass Spectrometry. 

Analytics

Considerable efforts have been made over the years to resolve the key issues of stability and delivery of RNA-based therapeutics.

The Marriage of RNA and Mass Spectrometry

The bio/pharma outsourcing industry continues to be integral in the development and manufacture of medicines. Contract research organizations, contract development and manufacturing organizations (CDMOs), and contract manufacturing organizations assist drug sponsor companies in bringing their products to market. These contractors offer the experience and resources some drug companies may lack to develop and manufacture drug products.

Outsourcing processes appears to be on the rise. When it comes to biopharmaceuticals specifically, BioPlan Associates states that more than half of biomanufacturers (65%) outsourced bioprocessing tasks in 2022, which is up 23% from 2006. The research company also predicts this trend will continue, citing that 79% of respondents to a 2022 survey have indicated they will be outsourcing some of their operations by 2027 (1). Analytical testing and toxicity testing were the most typical outsourced activities in 2022, according to BioPlan (2). “As the industry has become more complex, with increasingly advanced technologies, such as cell and gene therapies, in some cases, the need for the skills available from specialized CDMOs is indispensable,” said Shriya Bhatkhande, research associate with BioPlan Associates (2).

According to Ashley Wentworth, senior director, Vault Quality at Veeva Systems, contract partnerships are also entering the quality control space, with some companies collaborating with contractors in quality management. “Others, too concerned about security and data access, shied away from this approach. This, however, will change,” Wentworth said (3). In addition, contractors bringing suppliers into quality management systems will make acceptance, sign off, and approval of products easier, according to Wentworth. “Over time, companies will develop distinct information-sharing strategies, training programs, and new processes tailored to each partner” (3).

These and other outsourcing trends, such as fill/finish for messenger RNA therapies and technology transfer, are discussed in BioPharm International's sister publication's, 

Pharmaceutical Technology’s® Bio/Pharma Outsourcing Innovation eBook, published in February 2023. The eBook can be found on PharmTech.com.

BioPlan Associates, Inc. 19th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production (19th annual edition, April 2022).

Bhatkhande, S. Biopharmaceutical Manufacturing Growth. Pharmaceutical Technology’s Bio/Pharma Outsourcing Innovation eBook, February 2023.

Wentworth, A. Forecasting Trends in Pharma Innovation. Pharmaceutical Technology Bio/Pharma Outsourcing Innovation eBook. February 2023.

When referring to this article, please cite it as Haigney, S. Outsourcing Still Vital. 

From the Editor

The bio/pharma outsourcing industry continues to be integral in the development and manufacture of medicines. 

Outsourcing Still Vital

Bio/pharmaceutical companies operate in an industry known for its rigorous, exacting standards; companies place significant, implicit trust in any outside party they involve in their supply chain. Consequently, one could be forgiven for thinking that outsourcing materials for biologics production, which by design are much more complex than their small-molecule counterparts, would be infrequent because of concerns regarding quality control.

However, biologics outsourcing is a major industry that is, in concert with the rising demand for biologics throughout the industry, poised for rapid growth. A market report from IQ4I Research & Consultancy forecasted that this niche is expected to grow at a double-digit compound annual growth rate from 2022 to 2030. Additionally, the authors anticipated the industry reaching a $106.7 billion valuation by 2030 (1).

Outside of the growing demand for biologics, one can point to various reasons for this rise. For one, building a new biopharmaceutical manufacturing facility takes, on average, 5–10 years before it is operational, and can cost up to $2 billion (2). For small-to-mid-sized organizations, this cost is already an untenable expense, especially for products that are still in early-to-mid development phases. That said, even companies who have their own manufacturing facilities tend to dabble in outsourcing, as it allows them to leverage a contract manufacturing and development organization’s (CDMO’s) existing knowledge and experience with biologics production—for many companies, this is uncharted waters. In this vein, outsourcing can ultimately smooth out production irregularities and reduce time-to-market.

This begs the question: what is the state of this emergent industry? 

 spoke with Kasper Møller, chief technical officer at AGC Biologics, a CDMO specializing in protein-based biologics and advanced therapies, to outline the keys to a successful outsourcing platform, explore ongoing trends in biologics outsourcing, and much more on the topic.

When speaking with Møller, the core mantra that he repeatedly stressed was that biologics CDMOs need to be flexible. Because outsourcing companies are working with various companies on wildly different biologics requiring often disparate approaches—for instance, the needs of mammalian-derived therapeutics are different than those of microbial-derived proteins—versatility must be built into the framework of one’s solutions.

“Flexibility is key to adding value to each project and within each modality, facility, and manufacturing line,” said Møller. “Longer-term planning for projects can help address [this problem], but potentially more important is the use of flexible technology systems. With single-use technology and materials, it is possible to change the configuration of a manufacturing line, exchange or add unit operations, and cover various scales of operation.”

In Møller’s case, AGC Biologics developed “the 6-Pack single-use manufacturing concept,” which helps vary batch sizes within the same manufacturing line. This is achieved through combining harvests of parallel 2000-L single-use bioreactors, allowing the company to run anywhere from 2000-L–12,000-L batches at a time. Varied batch sizes allow customers to scale at their own pace, providing more time to evaluate product demand.

Møller also noted that this flexibility should also extend manufacturing agreements. For instance, in cases where high capacity is needed but the timing is uncertain, customizing a deal to provide a fully dedicated line, a partially dedicated line, or a line based off of a “rolling forecast” where products can be exchanged and manufacturing slots moved via a forecasting mechanism (Møller likened the last one to commercial manufacturing), can be helpful for fulfilling customer expectations while maintaining peak operational efficiency.

While biologics come in many forms—vaccines, recombinant proteins, gene therapies, etc.—Møller emphatically stated that he believes protein therapeutics will continue to be the top dog for years to come. Monoclonal antibodies (mAbs) in particular, he stressed, are something that AGC is focused on supporting, which the company facilitated by expanding its existing facilities in Copenhagen, Denmark in 2021 (3). According to Møller, this investment doubled their mammalian single-use system capacity at the site, adding a new manufacturing line with eight 2000-L single-use bioreactors for mammalian protein projects, a new 500-L perfusion bioreactor, and two downstream purification lines. Similarly, the company acquired a commercial-grade large-scale 20,000-L mammalian protein manufacturing facility in Boulder, Colo., in 2020 (4).

However, while mAbs are the king of the hill, they are not without competition. A key area of growth that Møller identified was cell and gene therapies, particularly in North America. In 2022 alone, FDA approved five novel cell and gene therapies, including Zyntelgo (betibeglogene autotemcel), Hemgenix (etranacogene dezaparvovec), Skysona (lisocabtagene maraleucel), Adstiladrin (nadofaragene firadenovec), and Carvykti (ciltacabtagene autoleucel); these 2022 approvals comprise roughly one-fifth of the total number of FDA-approved cell and gene therapies to date (5).

Møller’s observations are mirrored by many others in the industry: in 

 annual survey of bio/pharma workers, approximately half of them felt the biggest opportunity in the entire industry lay in cell and gene therapies (6). As companies race to compete in this rapidly growing niche, biologics-oriented CDMOs such as AGC will move to accommodate them. In the case of AGC, one such decision the company made was the acquisition of a of an established Italian cell and gene company, MolMed, in 2020 (7), as well as a Colorado facility containing viral vector and cell therapy production and manufacturing capabilities in 2021 (8).

While the exact needs of any company are typically unique in some form or fashion, Møller noted that are two core themes he finds himself telling his potential partners. “Two of the biggest considerations AGC Biologics focuses on with drug developers of any size is reducing cost and process robustness,” he said. “As an outsourcing partner, these are key areas that we advise our customers to watch closely.”

While few skimp on the actual development of the treatment, according to Moller, companies often neglect the design of robust manufacturing processes and analytical methods in the early stages of development in an effort to reduce costs and hasten time-to-market. However, this short-term outlook can present problems down the line.

Regardless of a treatment’s effectiveness, if there isn’t a scalable manufacturing process associated with it, there will almost certainly be delays when the time comes for commercialization. By taking the time to consider the design of manufacturing processes early on, companies can ultimately save themselves time and money.

Biologics Outsourcing Global Market–Forecast to 2030

2. Longo N. Setting up a Pharmaceutical Manufacturing Supply Chain Is a Complex and Lengthy Process. 

3. AGC Biologics. AGC Biologics Announces the Groundbreaking of its New Facility at its Copenhagen, Denmark Site. Press Release, Aug. 20, 2021.

4. AGC Biologics. AGC Acquires Biopharmaceutical Commercial Facility in Colorado, USA. Press Release, June 2, 2020.

5. FDA. Approved Cellular and Gene Therapy Products. 

6. Playter G. Crossing the Same River Multiple Times. 

7. AGC Biologics. MolMed S.p.A Becomes AGC Biologics S.p.A Following the July Acquisition. Press Release, Oct. 27, 2020. 

8. AGC Biologics. AGC Biologics Enters Agreement to Acquire Facility in Longmont, CO, Eyeing to Significantly Expand Their Cell and Gene Therapy Capabilities and Offerings. Press Release, July 1, 2021.


Vol. 36, No. 3
March 2023
Pages: 30–36

The trends shaping the growth of the biologics outsourcing industry demand attention.

BioPharm International, March 2023

Achieving High Yields in Upstream Processing of Plasmids

High-titer Vector Producing Cells

Tactics and Strategies for Designing an Ideal Lentiviral Vector Platform

Automated Inspection of Pre-filled Syringes and Biologics During Fill/Finish

The Marriage of RNA and Mass Spectrometry

Outsourcing Still Vital

The Logistics of Outsourcing Biologics

Vaccine Development Builds on COVID-19 Breakthroughs

Securing the Supply Chain with Good Distribution Practices

Subsurface Currents