Process Analytical Technologies for Manufacturing Cell and Gene Therapies

Defining cell and virus quality is the heart of successful cell and gene therapy production. Process analytical technology (PAT) that enables real-time monitoring and feedback control of manufacturing processes has the potential to increase productivity and product quality. Some solutions are currently available and being applied in the industry, but they do have limitations.

Greater process and product understanding and advances in fundamental technologies will be required before widespread application of PAT occurs for cell- and gene-therapy manufacturing. The excitement around these novel medicines will likely, however, help drive that needed innovation.

Numerous benefits of real-time monitoring and feedback control

Real-time monitoring and feedback control for cell- and gene-therapy manufacturing would allow real-time decision-making, reduce processing bottlenecks, and enhance process reproducibility and batch-to-batch comparability, according to Inbar Friedrich Ben-Nun, associate director of cell therapy R&D at Lonza. Making time-sensitive information available at a point where forward manufacturing decisions can still be made increases the value of process data, adds Richard Harrison, collaborations manager with Cell and Gene Therapy Catapult.

Reduction of manual process interventions would also result in decreased batch variability and/or failure, and lower the cost of manufacturing by reducing the use of various offline equipment and labor for analysis, Friedrich Ben-Nun says. Reductions in costs could also be realized through intensification of processes into smaller footprints and a reduction in number of operators required to run processes, while earlier alerts when failure does occur would help minimize lost product and the allocation of resources to failed batches, observes Harrison.

In addition to the development and implementation of robust and consistent manufacturing processes, real-time monitoring and feedback control enable a more flexible, tunable approach to manufacturing rather than a “one-size-fits-all” strategy, comments Pratik Jaluria, global head of technical development for cell therapies at Novartis. That would be particularly beneficial with respect to affording higher yields and accounting for donor-to-donor variability with autologous therapies, John Churchwell, senior scientist, Technology and Process Innovation at Cell and Gene Therapy Catapult, remarks.

Deeper insights into key inputs/outputs that could be predictive of clinical efficacy and safety would also be enabled by real-time monitoring and feedback control, according to Jaluria. Reductions in analysis times, meanwhile, would shorten the overall product release timeline, says Francesca Rossetti, analytical methods development manager with AGC Biologics’ Milan Cell & Gene Therapy Center of Excellence. “Real-time release of products,” concludes Churchwell, “would save time and money and get treatments to patients faster.”

Identifying the right parameters

The application of a quality-by-design (QbD) approach and PAT for real-time monitoring and feedback control for advanced therapy
medicinal product (ATMP)-manufacturing first requires the identification of relevant critical process parameters (CPPs), which can be challenging. “While many critical quality attributes (CQAs) are intuitive, such as quality, purity, and potency for cell therapies and additionally titer, infectivity, and empty/full capsid ratios for viral vectors, the process parameters that relate to these attributes are less obvious,” Churchwell explains.

“An effective approach is to investigate these correlations using a biomarker identification strategy that involves applying bioinformatic and systems-biology tools such as pathway analysis and canonical correlation analysis,” Churchwell believes.

Targeting upstream control for cell therapies

Cell quantity and quality are critical for the efficacy of cell-therapy applications and patient safety. Current cell-therapy manufacturing processes are, however, relatively fragmented, according to Brian Hassell, founder and CTO of Nirrin Technologies. “The goal for future PAT solutions, therefore, will be to connect all of the pieces of the cell therapy process,” he states.

Upstream cell-therapy processes, says Friedrich Ben-Nun, are usually continuous and involve long culture periods that allow for cell activation, cell expansion, and cell maturation or differentiation. Cell density can impact many aspects of culture processes, from gas exchange to the frequency of media replacement. Changes in the process, meanwhile, can affect cell viability and performance.

“For these reasons, it would be ideal during upstream processes to monitor cell density, cell viability, nutrients/metabolites in the culture media, growth factors in the media, cell performance (i.e., cytokine secretion), cell identity (via cell-surface markers or genetic markers), and bioburden,” Friedrich Ben-Nun comments.

Downstream processing of cell therapies, however, is generally different than that for traditional biologics. Cells are not commonly purified through a traditional chromatography column, though magnetic columns can be used with soluble components being added first to the cells, Jaluria explains.

“Considering the short term of the downstream process for cell therapies, it might be redundant or plethoric to apply PAT solutions, especially if they have not been adopted by regulatory authorities as substitutes for traditional cell-based assays. Nevertheless, this situation can lead to at-risk processing until the desired parameter can be measured,” Friedrich Ben-Nun says.

Jaluria adds that because downstream processing for cell therapies is really more about cell washing and buffer exchange, fill/finish technology equipped with sensors enabling real-time monitoring of specific quality attributes would be highly sought after.

Ideally, Jaluria observes, the impact of each unit operation on cell recovery including desired populations, cellular metabolic states, and other cellular properties would be evaluated. “In general,” Hassell concludes, “we need to be able to turn the knobs on transduction and expansion culture parameters while observing quantitative readouts for cellular identity, quantity and quality/function and have similar capabilities for downstream to ensure that final purification, formulation and storage do not affect any of the aforementioned attributes before administration of the product to the patient.”

Additional virus-related monitoring needed for viral therapies

While for viral vectors the cells are not the therapeutic product, many of the same process parameters need to be monitored during upstream processing (e.g., transfection or infection of cells). Additional upstream CQAs that should be monitored include viral particle production and packaging (full/partial/empty capsids), according to Friedrich Ben-Nun. Rossetti agrees that real-time monitoring would also be useful in viral particles testing to determine the best timing for vector harvest.

During downstream processing, Friedrich Ben-Nun says there is a need to continuously monitor the process, verify the viral product concentration, test for bioburden, identify empty vs. full capsids, and measure aggregates and other process-related impurities. “Particle aggregation,” stresses Rossetti, “is a very important parameter that should be monitored in real-time in order to reduce the risk of a low filtration yield.”

In general, Jaluria believes that PAT can aid in reducing the batch-to-batch variability commonly observed in current viral-vector manufacturing processes, which can impact a host of outputs including yield and various characteristics associated with release testing. “The implementation of PAT during all stages of production can improve process efficiency and increase product quality and safety,” Friedrich Ben-Nun confirms.

Application of PAT for cell and gene therapy production in its infancy

At present, only a few PAT solutions are being implemented for cell- and gene-therapy manufacturing. Many are the same as those used for cell-culture processes for traditional biologics, including sensors for monitoring dissolved oxygen, pH, temperature, and other basic process parameters. Most analyses for CQAs, however, are still performed offline, limiting their value for enhancing process control, Hassell states.

Enzymatic patch sensors for glucose/pH and basic capacitance-based pH probes are probably the most widely used PAT solutions today, observes Harrison. For more complex analytical monitoring, he notes that Raman spectroscopy represents the most mature of the complex spectroscopic PAT tools. Overall, though, Churchwell agrees that in the public domain there is limited application of advanced PAT in upstream ATMP manufacture and PAT in cell- and gene-therapy manufacturing is in its infancy.

The application of advanced PAT techniques such as Raman and 2D fluorescence spectroscopy have been shown, though, to enable real-time monitoring of a large number of additional parameters, Churchwell states. Examples include glucose, lactate, glutamine, glutamate, ammonia, total and viable cell density (TCD/VCD), product titer, cofactors, vitamins, and amino acids. Capacitance/dielectric spectroscopy also allows the direct monitoring of viable biomass, he says.

Specifically for some viral vectors, Jaluria adds that in-process sizing (i.e., cell size changes) and visualization are commonly used for upstream monitoring, with technologies that evaluate particle formation (charge-detection mass spectrometry, cryoelectron microscopy, and reverse-phase high performance liquid chromatography) increasingly being applied to intermediates.

During downstream purification, Friedrich Ben-Nun points to in-line pressure measurement during depth filtration as a PAT solution useful for indicating clogging for both cell therapies and viral vectors. She also notes that inline pressure and conductivity measurements are often collected during tangential filtration used for the harvest and formulation steps, while inline pressure, pH, conductivity, and UV absorbance monitoring are implemented during column chromatography for viral vectors.

Multi-angle light scattering (MALS), notes Churchwell, allows determination of the molecular weight of adeno-associated virus (AAV) vectors and quantification of the empty/full capsid ratio in downstream purification.

Current limitations

There are many reasons why PAT has not yet been widely implemented for cell- and gene-therapy manufacturing. At a fundamental level, there remains limited access to suitable technologies due to the need for advances in analytical technologies. Expertise in PAT is also needed, as in many instances existing processes/workflows need to be significantly modified before PAT can be implemented, according to Jaluria.

“The most widely in-process measurements only provide very basic process information. While the more complex spectroscopic PAT solutions such as Raman and other spectral techniques can give increasingly rich datasets, they are harder to robustly implement into a process and require dedicated expertise and experimental discovery runs in order to achieve performance at the required level,” Churchwell elucidates. “All advanced PATs have strengths and weaknesses; there is often a trade-off between sensitivity and chemical specificity,” he adds.

Robustness is also essential, stresses Hassell, because every company runs processes differently. Achieving a sufficient dynamic range for PAT sensors is an incredibly difficult problem to solve, however, primarily because processes can vary by two to three orders of magnitude, and manipulation of samples (concentration, dilution) introduces error. “For effective PAT, sensors need to be able to adapt to dynamic range and work across all scenarios,” he insists.

The complexity of cell- and gene-therapy manufacturing processes is also an important factor, according to Friedrich Ben-Nun. Measuring key factors in culture media, such as small molecules and growth factors, is generally not possible with most current PAT solutions. The complexity of the raw materials used in these processes can also hinder adoption of current PAT techniques, she says. In addition, the need for a high level of product understanding necessitates multiple experiments, with any process change requiring repetition of that work. The result, at least initially, can be manufacturing delays and/or deviations, which can put the process and product at risk.

More product understanding, better sampling, and challenges

The need to gain better product and process understanding—by running many experiments to enable linkage of product CQAs to CPPs—is, according to Friedrich Ben-Nun, an important challenge facing the implementation of PAT for cell- and gene-therapy manufacturing. Another is the large number of unit operations involved in some manufacturing processes, which means large numbers of CPPs that must be measured and higher capital investment associated with PAT implementation.

In addition, while regulatory agencies are encouraging the use of PAT for traditional biologic processing and would likely be accepted or even encourage PAT for cell-
and gene-therapy manufacturing, Friedrich Ben-Nun observes that it is not clear if PAT solutions could replace current offline bio-assays to determine, for instance, cell performance and bioburden levels.

There are fundamental technical challenges as well. “Much of the available tools were not explicitly designed for cell- and gene-therapy applications and may need re-designing or qualification prior to use,” explains Churchwell. “We are therefore likely to see a number of new devices tailored for this market emerging over the coming years that allow the connection of relevant advanced PAT solutions,” he adds.

For cell therapies, Jaluria points to the need to get a meaningful measurement of cellular properties quickly and in such a way that the material is not changing during sample preparation and manipulation so that the result is representative of the bulk that continues to be processed. “Reliable inline sensors that can provide release attributes in real time have yet to be developed,” he says.

For viral vectors, there are challenges around the small particle size of these materials, some of which can only be quantified using molecular methods, such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) techniques, according to Jaluria.

Sample size is also an issue. PAT methods must be developed that are highly sensitive and require only very small volumes given that process intermediates are precious materials. Instrument capabilities, Hassell remarks, need to accommodate both small samples and wide dynamic ranges.

In that vein, instruments must also be able to work with minimal
calibration and still give accurate measurements, particularly when PAT is being applied to autologous therapies. “All patients are different, and there is tremendous variability in processes and products, so robustness is essential across unknown scenarios,” underscores Hassell. Standardization of integration across scales and
platforms and wide acceptance of reliable scale-down models for developing and implementing PAT solutions are needed.

The lack of organizational awareness of the QbD methodology as applied to ATMPs and incorporating PAT to enable digitized manufacturing is another great challenge, according to Churchwell. “There is limited understanding of automation, physical techniques, and data-analysis/statistical-modeling methodologies. Implementing PAT solutions requires an integrated approach involving biomarker/CQA identification, PAT sensor application and the concomitant application of design-of-experiment approaches,” he explains.

Rossetti adds that the complex and time-consuming nature of most assays used for the characterization of ATMPs is a significant hurdle to the development of effective PAT solutions. “Many of these assays cannot easily be performed in a manner that generates results in real-time,” she explains. At AGC Biologics, to help reduce these challenges, the company developed a custom analytical platform. Optimization of the number and volume of samples required for quality control allows for faster turnaround times and reduced product consumption.

An industry-wide and collaborative effort to advance solutions

The interdisciplinary nature of cell and gene therapies has led to collaboration on research and development efforts spanning the fields of physics, chemistry, cell biology, engineering, computer science, and many others.

Greater dialogue between health authorities and drug developers around the numerous challenges facing cell and gene therapy manufacturing could also lay the foundation for how new technologies can be assessed and implemented, Jaluria believes. “One potential outcome of this interaction could be the preparation of general guidelines in which national or international agencies provide avenues and best practices around novel analytical technologies, progressing the field,” he says.

Industry-wide collaborations such as the National Institute for Innovation in Manufacturing Biopharmaceuticals, BioPhorum Cell & Gene Therapy, and Cell and Gene Therapy Catapult are, meanwhile, allowing companies to partner in advancing solutions.

As one specific example, Cell and Gene Therapy Catapult formed the PAT consortium to accelerate technology development and potentially lower the cost of cell-and gene-therapy manufacturing. This consortium will build industry awareness and leverage relevant skillsets to enable more rapid uptake of PAT technologies and digitized automation strategies and sensor integration. The consortium comprises more than 20 organizations ranging from pharmaceutical companies and technology providers to therapy developers and charities.

One goal of the PAT consortium is to act as a catalyst in accelerating the development of the necessary knowledge and understanding at reduced cost and investment risk to each organization.

“By reducing the barrier to entry for these technologies, we hope that more companies are able to take advantage of these techniques,” notes Harrison.The PAT consortium is the first large-scale dissemination activity in this field, but other “exciting future collaborations” can be expected. “With these efforts and the efforts of those across the industry, we expect to see an increase in the number of vendors supplying cell- and gene-therapy-specific instrumentation and a reduction in the barrier to entry for all cell- and gene-therapy companies to take advantage of these techniques in their processes,” Harrison ventures.

Active research area

Companies such as Lonza and Novartis are constantly evaluating potential new technologies that may overcome the challenges facing the industrialization of cell and gene therapies. “PAT is an active area of research, and a number of companies are looking to introduce their solutions in the next few years that could address many of the current bottlenecks,” Jaluria observes.

Rossetti points to the use of microfluidic systems to perform automated protein analyses (ELISA, Western Blot) as solutions that would allow operators to analyze more samples in the same test run, reducing time and cost. She cautions, though, that while these new and automated technologies show promising features, their development must be further advanced to ensure compliance with GMP regulatory requirements.

Nirrin Technologies is focused on developing PAT solutions based on near infrared (NIR) technology that address specific industry issues. “We use NIR optical sensors combined with machine learning to enable the next generation of upstream and downstream sensor solutions,” Hassell explains. He notes that the company is focused on the largest unmet needs, including easy-to-use, robust, accurate measurements for glucose, VCD/TCD and titer, with an emphasis on control system integration using the DeltaV Distributed Control System from Emerson.

Hopes for true real-time monitoring

Ultimately, the goal is to have real-time monitoring methods that can be implemented both upstream and downstream that require minimal sample volumes, enable comprehensive characterization of nucleic acids (RNA, DNA) and protein targets, and can be paired with control software for unit operations that allows for easy data analysis.

“At the deepest level,” states Hassell, “we want sufficient -omics content for understanding cell function, identity, and quality. For example, proteomics and transcriptomics combined with cytokine/chemokine analysis. Combined with data on more macroscopic properties such as cell number quantitation, viral titer, and full-versus-empty status for viral vectors, this type of solution would support the ultimate manufacturing platform.”

At Cell and Gene Therapy Catapult, the goal is to identify novel techniques and/or techniques that are new to the field that can address in-process measurement challenges in the industry. “In this respect, we are agnostic as to the specific methods employed, provided they help us measure identified CPPs and CQAs,” Churchwell comments.

Specific PAT solutions that Friedrich Ben-Nun would like to see realized include a rapid real-time method to monitor growth factors during cell production; a real-time method to identify cell-type impurities such as immature or undifferentiated cells during manufacturing; a real-time method for detecting cell characteristic changes (cell shape, nucleus-to-cytoplasm ratio, hormone secretion, etc.) as an indicator of process success; a real-time method for monitoring virus particle number, aggregation, and genomic content; biomarker-based methods for correlating or predicting product potency; a rapid real-time method to identify impurities during viral-vector purification; and incorporation of automated sample systems with targeted analytical solutions.

For vector production processes, Rossetti says that rapid ELISA assay testing with microfluidic technology for quantification of the p24 capsid protein could help to immediately understand the production yield and decide if a team should proceed with downstream processing. She also notes that real-time analysis of process-related contaminates during downstream processing would also be valuable. For cell therapies, Rosetti would also appreciate a PAT system that can analyze not only viability, but immunophenotype as well, to better characterize cell subpopulations and evaluate the level of differentiation for products that are ultimately being infused into patients.

Ideal timing

As the field continues to mature, cell- and gene-therapy manufacturing are perfectly positioned to benefit from advances in PAT technology developed for mammalian manufacturing of traditional biologics. “Fine-tuning and optimization of the tools developed for mammalian manufacturing of biologics will be critical to enable the adoption of PAT for cell- and gene-therapy manufacturing,” Friedrich Ben-Nun says. She adds, though, that new PAT solutions with specific applications to cell- and gene-therapy manufacturing will need to be developed in order to support the variable process pipeline in cell and gene therapy.

Jaluria agrees that there are great opportunities in the space of inline and nondestructive analytics for use in the production of cell and gene therapies. Hassell goes even further: “The timing is ideal for PAT in cell and gene therapy. It will start to succeed because of the immense amount of research and excitement surrounding ATMPs such as cell and gene therapies, which is driving the development of novel, next-generation biological sensors.”

About the author

Cynthia A. Challener, PhD is contributing editor to BioPharm International.

Article Details

BioPharm International
Vol. 34, No. 12
December 2021
Pages: 10–14

Citation

When referring to this article, please cite it as C. Challener, “Process Analytical Technologies for Manufacturing Cell and Gene Therapies,” BioPharm International 34 (12) 10–14 (2021).