Mammalian cell lines, particularly Chinese hamster ovary (CHO) cell lines, have become the standard expression systems for the production of biologic drug substances from recombinant proteins to more complex monoclonal antibodies (mAbs). CHO cells have played a significant role in the manufacture of revolutionary drugs for the treatment of many diseases, and their use is still the focus of major investment among biopharma companies worldwide, according to Barry Holtz, president of iBio. Although the standard, they do possess limitations that need to be addressed as the biopharmaceutical industry evolves to meet government, payer, and patient expectations for cost-effective, safe, and efficacious medicines. In addition, conventional mammalian cell lines may be inappropriate for the production of next-generation medicines such as bi/multi-specific antibodies and gene and cell therapies.

	High cost and long development timelines are two major drawbacks of conventional expression systems, according to Mike Laird, senior director and principal scientist for process development at Genentech. These systems also have the potential for low expression levels for some novel protein structures.

	The biggest issue, asserts Holtz, is the time involved in the development of mammalian expression systems. “Traditional mammalian cell-based systems require the development of stable cell lines that perform well, which requires the completion of multiple evaluations in small reactors over several months. While the product is well-characterized at that point, scale up to larger reactors is often needed, and the environmental changes in bigger vessels, whether single use or stainless steel, can impact the post-translational modification of the protein, which can cause problems and delays in the business timeline. At each step, the protein must be extensively characterized to assure efficacy and potency. All of this effort adds significant expense as well.”

	Therapeutic protein production using CHO is expensive, agrees Mark Emalfarb, founder, CEO, president, and director of Dyadic International. “Mammalian expression systems require costly upfront investments in manufacturing facilities and high material and production costs. In addition, CHO expression entails a relatively low mAb yield (low-single-digit g/L/d) and a long cycle time. Furthermore, CHO cell lines typically require two viral purification steps, which are not necessary for some alternative systems, such as the 

 (C1) fungal system we are developing. C1 has no viruses and thus the need for those purification steps is eliminated,” he observes.

	Another important issue relates to the fact that the current generation of mammalian expression systems has not seen the complex, non-natural protein formats currently found in discovery and thus their synthesis, folding, and secretion machinery has not evolved to handle such proteins, according to Andy Racher, associate director of future technologies at Lonza Pharma & Biotech. “These systems have limited ability to produce these new proteins with clinically relevant attributes and in clinically relevant amounts,” he notes. In addition, many new proteins contain three or four rather than one or two different polypeptides, and current expression vector formats are challenged by these new protein heteromers.

	“It is time to realize the limitations of CHO and look beyond it to explore newer and potentially more efficient drug development and production methods,” asserts Emalfarb.

	As the need to make biologic drugs more accessible and affordable to patients increases, the industry is ramping up its investigation of other manufacturing methods. The drawbacks of mammalian expression systems are also driving the exploration of new and alternative technologies to move many next-generation biologic drug candidates through later development stages.

	A number of “new” technologies will become more routine as the demands for new “designed” proteins strain the capabilities of conventional CHO cell systems, according to Holtz. One important approach is the engineering of new mammalian cell lines using new genetic editing technologies and the innovative design of gene constructs to optimize yields. He also notes that techniques to evaluate libraries of cell lines will help optimize expression and yield.

	Lonza, for instance, has developed a suite of multigene vectors where three, four, and possibly more different genes can be easily inserted into a single expression vector. “By putting all the genes into a single vector, all genes are ensured of being inserted into a transcriptionally active locus in the genome and being transcribed at high levels,” Racher says.

	There are also efforts to develop entirely different expression systems based on plants, baculovirus, bacteria (such as 

 in the Phoenix system), and yeast, many of which have already been demonstrated to get proteins to the clinic and licensure, according to Holtz.

	“In the not-too-distant future, it is likely that drug companies will evaluate two or more expression systems simultaneously as a routine best-practices approach in early stage development,” he comments.

	iBio’s plant-based system offers rapid evaluation of protein expression at a very low cost, according to Holtz. Because vectors are used to transfect the plant leaf cells, multiple constructs can be evaluated in parallel. Once infected, the plants produce the required proteins in less than seven days. At that point they are harvested, homogenized, and a clarified protein extract is ready for traditional protein separation and purification. In addition, scale-up is seamless and reproducible; each 10-g plant is an individual bioreactor, so it is only a matter of growing more plants and there are no issues around changes in protein structure or posttranslational modification, according to Holtz. He also notes that plant bioreactors are grown with no human or animal-derived materials and are not handled at any time by humans, which eliminates the chance that mammalian adventitious viruses will be present.

	Production of plant-made biologics been scaled-up by several companies in new facilities that can produce hundreds of kilos of mAbs and other therapeutic proteins per year. Successful antibodies (cancer vaccines and others) and other therapeutic proteins such as enzyme replacement therapies have been successfully taken to advanced clinical trials and some to licensure. In all cases, there have been no reports of adverse events associated with production of therapies in plants,” Holtz states.

	iBio grows 2.2 million plants continuously at its Texas facility and has worked with a variety of clients to produce mAbs, fusion proteins, antibody-drug conjugates, and vaccines, including virus-like particles (VLPs). “We have invested in increased product and process facilities and a cGMP-compliant pilot plant that-coupled with our large-scale manufacturing facility-assures clients that they can develop their protein through clinical trials and then be supported for the commercial launch of their products,” says Holtz. iBio will also transfer the technology to clients if they want to build and operate their own facilities.

	Emalfarb believes that the C1 fungal expression system may one day be a safe and efficient approach to speeding up the development, lowering the production costs, and improving the performance of vaccines and biologic drugs at flexible commercial scales.

	Dyadic’s Cl gene expression platform is based on technology originally developed for industrial biotech applications, such as biofuel and enzyme production, and sold to DuPont for $75 million in December 2015. The genetically modified strain of 

 is designed to produce enzymes and other proteins at a rapid rate. The company retained the rights to apply C1 to human and animal biopharma applications and has been investigating its use for the production of mAbs with humanized glycostructures; non-glycosylated mAbs, antibody fragments, FC fusion proteins, next-generation biologics, and other therapeutic proteins for which glycosylation structures are undesirable; and antigens, vaccines, and VLPs.

	“We are applying the power of an industrially proven gene-expression system that has been used by the likes of Abengoa, BASF, Dyadic, DuPont, and Shell Oil, among others, to produce industrial enzymes and proteins at greater than 100 g/L of total protein at up to 80% purity (80 g/L) at commercial scales greater by 25 times (500,000-L scale) or more than some of the largest CHO bioreactors (12,000-L scale) in one-half to one-third the time,” Emalfarb explains, noting that there is still room for yield improvement with C1. He adds that Dyadic has to date achieved a productivity for mAbs of 9 g/L in 90 hours or a 2.4 g/L/d production rate, which can be compared to 4 g/L in 336 hours or a 0.30 g/L/d for typical CHO processes, an eight-fold improvement.

	Emalfarb notes that the media cost for C1 is a fraction of that for CHO, there is no need for viral inactivation, and C1-expressed proteins are secreted from the cells in a purer form than those produced by CHO cells so are likely to be quicker and easier to purify.

	Dyadic is currently working with pharmaceutical companies that are researching its C1 platform to speed up the development and lower the cost of biologics, enable the development and commercialization of genes that are difficult to express at reasonable yields in CHO and other cell lines, and apply C1 for the production of larger quantities of proteins earlier in discovery and development. The company and its partners are also investigating the possibility of getting difficult-to-express genes that have potential as new and novel cures-but have been shelved due to lack of expression into the clinic-in a commercializable and affordable way, according to Emalfarb.

	In late 2017, Lonza introduced a new yeast-based expression system for the production of next-generation biologics. Its XS Pichia 2.0 Expression and Manufacturing Platform, based on 

, was designed to combine the best features of bacterial and mammalian systems in one system: fast and easy strain development and robust and rapid fermentation combined with a highly pure secreted product for a simple downstream processing, according to Christoph Kiziak, research and technology lead for microbial technology at Lonza Pharma & Biotech.

	“The driving force was to rethink the whole production strategy for producing proteins to circumvent the main bottlenecks of bacterial systems (e.g., intracellular production, endotoxin), CHO systems (e.g., time-consuming, viral clearance), and yeast systems (e.g., use of methanol, hyperglycosylation), while maintaining the use of 

 due to the general advantages and regulatory acceptance of this yeast cell,” Kiziak says.

	The “auto-inducible” setup of the new system makes it convenient for high-throughput clone screening, which results in highly pure material for preliminary quality analysis of the product at an early time point.

	In addition, fermentation development follows a product-specific model-based approach, which allows yeast fermentations to be performed in two to three days with a high volumetric productivity, according to Kiziak. It can also be expanded by an 

 model for process productivities over a wide production window. “This predictive model allows us to take into account production plant and process-specific limitations at any stage of development and provides high flexibility and quality for later production,” he explains.

	The methanol-free process also avoids the negative impact on cell viability and product quality of the commonly used AOX1 system, according to Kiziak. There is no need for explosion-proof facilities and the lower oxygen transfer rates compared to the AOX1 system provide additional flexibility regarding production plant requirements. There is also no need for endotoxin or viral clearance testing.

	Furthermore, the product is secreted into the culture supernatant, where the minimal medium together with the low host-cell protein background provides an ideal starting point for an efficient downstream process.

	To date, Lonza has focused on producing multi-specific novel antibody mimetics from various sources using the XS Pichia 2.0 and has achieved productivities of more than 2 g/L per day. The company is working to make the system even more customer friendly and easy to apply and to refine the model-based approach in order to improve the accuracy of the prediction of fermentation processes. Additional promoters with different strengths and induction profiles are also being developed to allow the tuned expression of helper factors, auxiliary proteins, heteromeric products, enzyme cascades, etc., which will expand the applicability of the XS Pichia 2.0 in the future, according to Kiziak.

	Regardless of the technology, there is a general acceptance that existing mammalian expression technology can no longer meet the needs of the biopharmaceutical industry. “One way to make healthcare more accessible and affordable to patients could be changing the cell lines we use for manufacturing,” Emalfarb observes. “Our goal is to bring affordable medicines to more patients, in addition to improving processes to develop new treatments,” he adds.

	The industry isn’t there just yet, however, according to Laird. “At this time, we are not aware of novel expression technologies appropriate for commercialization of next-gen biologics with significantly reduced costs, timelines, or complexity that can also ensure consistent post-translational modifications such as glycosylation. Although some new complex molecules could be harder to express using current or conventional mammalian expression systems, we think these systems are and will be the best approaches to express proteins for therapeutic purposes for the foreseeable future, especially given the vast knowledge from current advances in genome sequencing and CRISPR [clustered regularly interspaced short palindromic repeats] gene-editing technology that can be used to modify these conventional mammalian expression systems,” he explains.

	“With that said, we are very open to evaluating novel expression systems and will feverishly pursue new technologies as they become available,” Laird asserts. “We all have the same goal of delivering medicines to patients as quickly and efficiently as possible,” he concludes.

	Genentech is currently focused on the development of targeted integration mammalian cell lines to enable faster, more consistent medicine development with higher productivities. To date, engineered host-cell lines to optimize performance, increase productivity, ensure product quality, increase the ability to produce complex formats, and decrease timelines have been achieved, according to Laird.


	Vol. 31, No. 8
	August 2018
	Page: 16–19

	When referring to this article, please cite it as C. Challener, “The Search for Next-Gen Expression Systems" 

Articles

Features

Biopharma seeks alternatives that meet the needs for next-gen biologic drug production.

The Search for Next-Gen Expression Systems

	While many people headed to summer vacation fun, FDA was busy in July, including a reorganization plan, new guidance documents for gene therapy development, efforts to prevent drug shortages, and more flexible import policies.

	FDA’s Biosimilars Action Plan (1), announced on July 18, 2018, may have the most implications for the market segment. In introducing the plan, FDA Commissioner Scott Gottlieb took innovator biopharmaceutical companies to task for impeding the delivery of biosimilar drugs to market (2). To date, only three of the 11 biosimilar drugs approved by FDA have reached patients, and the lack of competition in the biosimilar space cost US patients more than $4.5 billion in 2017, Gottlieb noted.

	“Sometimes it feels as if we’re seeing the biosimilars version of ‘Groundhog Day,’ with brand drug makers replaying many of the same tactics, and all of us being too susceptible to many of the same misconceptions about biosimilars’ safety and efficacy relative to originator biologics,” Gottlieb said in prepared remarks at the Brookings Institution. “We’re falling into some of the same doubts and policy constraints that were used to deter competition from generics in the years after the Hatch Waxman Act. But we’re not going to play regulatory whack-a-mole with companies trying to unfairly delay or derail the entry of biosimilar competitors. We’re not going to wait a decade or more for robust biosimilar competition to emerge.”

	Delaying tactics may discourage biosimilar sponsors from developing products and reduce public confidence in market-based pricing mechanisms, ultimately hurting innovator companies, Gottlieb said; therefore, FDA’s Biosimilars Action Plan seeks to achieve a balance between innovation and competition. The Plan, part of the Trump Administration’s Blueprint to Lower Drug Prices, focuses on improving the efficiency of the biosimilar and interchangeable product development and approval process; maximizing scientific and regulatory clarity for biosimilar product development; improving understanding of biosimilars among patients, clinicians, and payors; and supporting market competition by reducing gaming of FDA requirements or other attempts to unfairly delay competition.

	“Effective market competition from biosimilars depends on additional actions from our public and private sector partners to align reimbursement and formulary design to encourage appropriate biosimilar adoption,” Gottlieb said. “Competition requires all of us to shine a light on the anti-competitive impact of tying rebates and bundling biologics with other products to protect biologics’ market share. And it requires us to educate providers and patients about biosimilars, and why people should have confidence in the safety and effectiveness of these FDA-approved products,” he said.

	Prior to the introduction of the Biosimilar Action Plan, the Biotechnology Innovation Organization said-in response to the Blueprint to Lower Drug Prices-that it was working to “increase marketplace competition by speeding regulatory approval of more innovative drugs, and promoting greater and faster generic and biosimilar entry once patents and exclusivities for innovator drugs have expired,” but opposed ideas that “impeded innovation” such as price controls, drug importation, or direct government of negotiation of Medicare drug prices (3).

	While there appears to be general agreement on the end-game, the means of getting there are up for debate. A public hearing scheduled for Sept. 4, 2018 could have some interesting conversations.

Biosimilars Action Plan: Balancing Innovation and Competition

Remarks from FDA Commissioner Scott Gottlieb, M.D., as prepared for delivery at the Brookings Institution on the release of the FDA’s Biosimilars Action Plan

, July 18, 2018.
	3. Biotechnology Innovation Organization, “

BIO Submits Comments Re: HHS Blueprint to Lower Drug Prices and Reduce Out-of-Pocket Costs

	When referring to this article, please cite it as R. Peters, " FDA Is Not Playing Games with Biosimilars" BioPharm International 31 (8) 2018.

From the Editor

Frustrated by slow market adoption, Gottlieb maps out new plan for biosimilar competition.

FDA Is Not Playing Games with Biosimilars

Process- and product-related impurities must be evaluated according to various regulatory guidelines during production and to enable final product release. Impurities can arise from the biological samples themselves or from the process of developing biologics, including handling of materials.

	Sample-related impurities include residual host cell-derived proteins (HCPs) and nucleic acids, complexes or aggregates of the biologic (high-molecular-weight [HMW] proteins), and clipped species and half molecules (low-molecular-weight [LMW] proteins). Impurities from cell-culture media can include inducers, antibiotics, and media components.

	Impurities that come from downstream processing can include microscopic particulates, metals, and any materials that have carried over from the purification process, including resin particles, surfactants, emulsifiers, and viral-inactivation agents. Biological contaminants derived from handling include mycoplasma, bacteria, and virus particles.

	Some of these impurities have known structures, while others may be only partially characterized or completely unknown. Post-translational modifications such as glycosylation and phosphorylation, degradation via oxidation or deamidation, and disulfide bridge scrambling (misfolding) can occur during upstream or downstream processing or storage under inappropriate conditions, resulting in large numbers of possible impurities. Disposable equipment and plastic tubing, stoppers, and containers may be sources of leachables. For antibody-drug conjugates, free drug cytotoxins can be problematic.

	The decision on whether to monitor these impurities, and to what levels, is generally risk-based, using knowledge from both analytical and biological assays, and any preclinical experience to assess the impacts of each impurity on the safety, efficacy, or stability of the biotherapeutic, according to Scott Berger, senior manager for biopharmaceutical markets at Waters Corporation.

	Monitoring biologic production processes and analyzing products for release testing can be challenging for many reasons. For Jean-Francois Boe, scientific director of SGS Life Sciences, the greatest challenge is the vast number of potential impurities that can be formed when all of the possible chemical modifications that can occur are considered. “Tens of millions of combinations of impurities can be formed, many of which have significantly different physical and chemical properties. One unique analytical technique cannot be used. A number of appropriate analytical methods must be used to create as full a picture as possible of the impurities that are present,” he explains.

	“Purification of biologics is often a multi-step process, and there is no one-size-fits-all analytical methodology,” adds Tiffani Manolis, director of global pharma segment marketing with Agilent Technologies.  “As a result, analysis of residual impurities is often a time-consuming activity.”

	Another major challenge when developing methods to evaluate bioprocess residuals is matrix interference, according to Jon S. Kauffman, president of Eurofins Advantar Laboratories, a member of Eurofins BioPharma Product Testing.

	“Developing a robust method for certain impurities is always a challenge. For most of the methods that support in-process or release testing of drug substances, both matrix effects and the presence of a high concentration of product are the main factors which can impact the performance of methods,” agrees Jun Lu, director of analytical development at Catalent Biologics.

	Matrix interference can be caused by components in the formulation buffer that interfere with the detection of the residual by suppressing the ionization in the mass spectrometer or from the residual binding to the protein, according to Kauffman. “Further,” he says, “we are typically required to monitor these residuals in various steps throughout the bioprocess. The sample matrices from each step can be quite different and each pose a challenge with respect to interferences and sample preparation.”

	Complicating the situation is the fact that many product-related impurities need to be monitored down to low-percentage, or fractional-percentage levels, straining traditional optical, ultraviolet (UV)-based peptide mapping assays, according to Berger. “Increasingly, this necessitates the use of liquid chromatography-mass spectrometry (LC-MS) analysis to obtain the additional the selectivity and dynamic range for detection and monitoring of critical impurities. In addition, some impurities such as clips and unfolded variants may require multiple techniques for efficient detection and quantification, because peptide level analysis is often uninformative for these structures,” he observes.

	Some impurities, such as surfactants, often exhibit a broad rather than a sharp peak and can interfere with each other, making specificity difficult to obtain. “For example,” notes Kauffman, “it is virtually impossible to detect poloxamer 188 in a drug substance/product that is formulated with polysorbate. In these instances, we are forced to go backward in the manufacturing process to the step prior to addition of polysorbate.”

	Other challenges include the need to derivatize LMW compounds before analysis, as well as the ability of some residuals to adhere to the surfaces used during sample preparation, and the instability of others. Understanding these possible issues when developing methods is extremely important, according to Kauffman.

	Numerous monitoring, separation, and detection technologies

	As biopharmaceutical production processes evolve, and with the complexity of new process matrices, the detection and tracking of residual impurities is becoming increasingly difficult and may require various orthogonal techniques, says Vincy Abraham, director of biologics, Catalent Biologics.

	Liquid chromatography and electrophoresis remain the two main separation techniques, and immunochemical assays remain unavoidable in specific cases for the evaluation of low levels of residual impurities, according to Boe. He notes that while little has changed with these separation technologies, there are many more advanced detection methods available today. UV or visible light and infrared (IR), fluorescence, mass spec, light scattering, and more have improved capabilities.

	Other separation methods include gel-permeation, size-exclusion chromatography, ion exchange chromatography, and gas chromatography. For Kauffman, LC-MS/MS performed using a triple-quad mass spectrometer connected to an ultra-high-pressure LC (UHPLC) system is the technique of choice given its sensitivity, specificity, and ability to provide quantitative results. “This instrumentation is required in most cases to be able to quantitate at the ng/mL or even pg/mL range at which residuals must be evaluated,” he says. HPLC and UHPLC are, however, still used with UV, charged aerosol, or evaporative light scattering detectors for compounds of interest in the µg/mL range or higher that do not ionize.

	Detection by mass spectrometry is particularly useful for evaluating residual impurities formed due to chemical modification of the biologic drug substance, according to Manolis. Depending on the specific species of interest, MS can be coupled with LC, gas chromatography, matrix-assisted laser desorption ionization (MALDI), and electrospray ionization (ESI).

	The most common method for screening biopharmaceutical products and testing for HCPs is enzyme-linked immunosorbent assay (ELISA), a sensitive assay with a low detection limit, high level of reproducibility, and compatibility with high-throughput screening, according to Laura Moriarty, marketing manager for Bio-Rad’s Drug Discovery and Development Group. She notes, though, that because the ELISA technique does not permit identification of antigens when using mixtures of antibodies, but only provides titers, the accuracy and utility of ELISA relies on a thorough prior assessment of the antibodies used. “Accurate evaluation and validation of antibodies reactive against HCP is crucial for detecting and monitoring HCP both during the product development cycle and during manufacture of biologics,” she says.

	The predominant method for assessing anti-host HCP antibodies involves 1-D or 2-D electrophoresis followed by western blotting, according to Moriarty. For polypeptides with similar molecular masses in complex mixtures of proteins, 2-D electrophoresis gives much better resolution because it separates proteins in the first dimension by isoelectric point (pI), followed by molecular mass in the second dimension. Once a good purification system has been established, the final product can be routinely screened with an ELISA to make sure that impurities are continually removed from the samples.

	For nucleic acid screening, quantitative polymerase chain reaction (qPCR) and droplet digital PCR are used to detect and signal the presence of nucleic acids in a sample. Mycoplasma can also be detected using PCR, as well as colometric enzyme assays. Bacteria can be detected using endotoxin testing via the limulus amebocyte lysate assay, the United States Pharmacopeia (USP) chromogenic method, and the gel-clot method. The types of viral strains to be tested are specific to the method used to manufacture a therapeutic or biological.

	Biologic aggregates are typically detected using sedimentation velocity analytical ultracentrifugation (SV-AUC), size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), or dynamic light scattering (DLS) for the analysis of quaternary structures. DLS, as well as resonant mass measurement (RMM), can also be used to detect microscopic particulates, according to Moriarty.

	Because there are so many different types of manufacturing processes and residual impurities from low to high molecular weight with varying chemical and physical properties, identifying multifunctional methods that can separate and detect more than one type of impurity is essential for developing optimized methods. “Mass spectrometry is becoming attractive in part for this reason; a mass spectrometer can be used for the detection of numerous different impurities well chromatographically separated or co-eluted in a single chromatographic run,” Boe states.

	Mass spectrometry has become the primary analytical technology applied to multiplexed analyte detection within complex samples, agrees Berger. “The additional selectivity of the mass dimension enables detection and higher dynamic range quantification of analytes, even in the presence of co-eluting species. This methodology is now starting to be applied within biopharmaceutical development against a list of targeted product or process impurities,” he observes.

	Recently there has been a lot of work done using LC–MS for multi-attribute monitoring method (MAM), which is designed, according to Manolis, to provide simultaneous detection, specificity, identification, quantitation, and monitoring of attributes that are relevant to safety, efficacy, and the overall quality of drug. “MAM provides residue-specific identification, quantitation, and better understanding of any post-translation modification when compared to traditional methods of analysis, thus improving overall operational efficiency, resource consumption, and time required,” she comments.

	As long as the transitions monitored are distinct for each compound with little to no cross talk, Kauffman agrees that newer LC–MS/MS systems and software suites allow the detection of multiple impurities at once. “The challenge in these situations is the sample prep. Often times when you optimize a method for multiple analytes, it works really well for some analytes but not for others. Finding the right sample prep that extracts all the analytes of interest can be quite challenging. Methods for sample cleanup often work for one sample matrix but not another. As a result, the rule of one analyte per method is still the preferred approach so that the method can be optimized for the analysis of that particular analyte,” he says.

	“With ever-increasing regulatory and compendial stringency to identify, quantify, and monitor impurities, a greater emphasis is being placed on their characterization and analysis at trace levels,” asserts Abraham. “Fortunately,” she continues, “there have been parallel advancement in technologies that allow rapid characterization of impurities at levels of approximately 0.1%.”

	To alleviate some of the limitations with ELISA, for instance, Abraham notes that several technologies for quantitation exemplified by Gyrolab, AlphaLISA, and Octet have emerged in the past decade as viable alternatives for HCP. Each represents a different strategy for HCP quantitation.

	Bio-Rad recently introduced droplet digital PCR (ddPCR) as a sensitive (picogram range sensitivity in milligrams of recombinant vaccines) and quantitative method for quantification of residual host-cell DNA, according to Madhuri Ganta, senior global product manager in Bio-Rad’s Digital Biology Group. With ddPCR, a sample is partitioned into 20,000 nanoliter-sized droplets, which makes the PCR reaction less susceptible to inhibitory substances. Unlike with qPCR, extraction of total DNA from the protein drug sample is not required; intermediates can be processed directly, and absolute quantification is possible without the need to establish a standard curve, according to Ganta.

	While optical-based LC assays are still highly desirable due to the lower system cost and broader organizational accessibility of this technology, Berger observes that the increasing complexity of modern biopharmaceuticals has pushed laboratories to adopt more resolving and sensitive UPLC- and UHPLC-based separations platforms for these newer products. He adds that the additional adoption of mass detection to increase selectivity and dynamic range of these assays has been growing within regulated development and is now starting to appear in quality control for targeted monitoring of product and process attributes and impurities.

	The use of mass spectrometry for the characterization and quantification of HCPs is an active area, according to Yunsong (Frank) Li, director of biologics process development at Catalent Biologics. “MS can detect the HCPs not covered by anti-HCP reagents and provide additional information such as molecular weight, theoretical isoelectric point (pI), and immunogenicity potential,” he explains. ProteinSEQ technology (Thermo Fisher Scientific) has also recently been demonstrated to quantify HCPs in a much wider dynamic range than ELISA, according to Li. The combination of ion exchange (IEX)-HPLC and high-throughput western blot is also under development for quantification of low immunoreactive HCPs.

	For detection of aggregates, Li adds that nanoparticle tracking analysis (NTA) can track nano-sized particles via particle-scattered light from a focused laser beam. “The system can track many individual particles and therefore count the number of particles. From the rate of the particles’ Brownian movement, the size can also be calculated,” he says. Flow cytometry, traditionally used for cell counting, has also been developed to count the protein aggregation particle size as low as 0.2 µm.

	In other areas, traditional sodium sodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) is being replaced by capillary electrophoresis (CE-SDS) because it provides superior detection, reproducibility, and robustness, according to Manolis.

	Another development, according to Abraham, involves a shift from the conventional protocol of isolation and spectral analysis to online analysis using sophisticated modern hyphenated tools, such as GC-MS, LC-MS, CE-MS, supercritical fluid chromatography-MS (SFC-MS), LC-nuclear magnetic resonance (LC-NMR), CE-NMR, and LC-Fourier-transform infrared spectrometry (LC-FTIR).

	Separately, Berger points out that the use of automation for sample preparation is greatly increasing within development and quality control organizations. “In development, this automation often supports higher-throughput clone selection and quality-by-design (QbD) studies, but increasingly the reason for adopting automated sample preparation is the improved consistency of sample generation versus manual workflows. The need for a mid-tier scale of automation has become apparent,” he says.

	Indeed, improving the efficiency and reducing the costs associated with residual impurity analysis, which is essential to improving the overall efficiency drug development and manufacturing, requires that workflows be amenable to automation and high-throughput protocols, agrees Moriarty.

	Eurofins is typically required to resolve three primary problems that are interconnected: quantitation limits, interfering compounds, and extraction of analytes of interest. “Interfering compounds and poor extraction of the compounds of interest directly affect the quantitation limits of the methods. Mass spectrometry for the most part eliminates co-eluting peaks because we can focus in on a mass transition for the compound of interest, but there are still times when compounds share the same transitions or have cross talk with transitions from other compounds. Extraction techniques have evolved over time especially with the addition of molecular weight cut-off filters and solid-phase extraction cartridges, but the more you manipulate the samples, the more chance you have to introduce error and contamination,” explains Kauffman.

	One challenge is the high variability in the process and sample matrix, which can contribute to out-of-specification/out-of-events, which are often time-consuming and costly, according to Manolis. Standardization of specifications for critical reagents and simplified and reproducible processes for sample digestion are also needed. For multi-impurity detection methods such as MAM, Manolis notes that improvements in systems for data processing, handling, and interpretation are needed.

	Boe points to the current gap in the ability to accurately characterize and mostly quantify particles (aggregates) that are between several hundred nanometers up to 1 micrometer in size. For HCPs, he notes that the need to switch from commercial kits for HCP analysis to custom-developed methods once a candidate reaches Phase III trials is time consuming.

	Currently, the greatest limitation for process-related impurity is analytical technology for HCP analysis, with the major challenge in coverage from existing anti-HCP polyclonal antisera standards, according to Li. The current approach is to develop product- and process-specific assays, which often require long lead times of at least 18 months, or combine multiple existing anti-HCP polyclonal antisera standards.

	A general key challenge has been increasing the usability of more informative and complex modern analytical technologies to enable non-specialists to continue to perform these analyses, according to Berger. “While those charged with product characterization are always welcoming greater performance envelopes of their instruments, those charged with product monitoring and release now tend to be focused on minimizing user interactions with their systems and maximizing quality and reproducibility of the results,” he says.

	In addition to establishing methods that meet requirements for sensitivity and specificity, there are other factors that are important to consider. “It is essential to first determine the appropriate acceptance criteria and then ensure that methods can be readily transferred from R&D to commercial production. They should be robust, accurate, and precise, as well as easy to implement on equipment that will be available at the manufacturing plant,” Boe asserts.

	A validation process that makes sense is also important, as is the need to consider the biological activity of product-related impurities. “Some impurities that are closely related to the product may have the same biologic activity as the drug substance, and therefore may not impact the safety and efficacy of the product. It may be reasonable to classify these compounds as related substances, rather than residual impurities,” Boe explains.

Vol. 31, No. 8
	August 2018
	Page: 20–25

	When referring to this article, please cite it as C. Challener, "Expectations for Residual Impurity Analysis Continue to Rise" 

More complex biologic samples must be evaluated to ever higher levels of specificity and sensitivity. 

Expectations for Residual Impurity Analysis Continue to Rise

The packaging or container closure system that is meant to protect a pharmaceutical product can be a source of contamination. Comprehensive extractables and leachables (E&L) studies are, therefore, required to identify and quantify harmful impurities that could affect the quality and safety of drug products.

spoke with Lester Taylor, Pharma marketing manager, Agilent Technologies; Andrew Blakinger, manager, Extractables and Leachables Testing, Eurofins Lancaster Laboratories; and Fran DeGrazio, vice-president, Global Scientific Affairs and Technical Services, West Pharmaceutical Services, about the ins and outs of extractables and leachables assessments in biologic drug products.  

 What are the E&L challenges for biologics compared to small-molecule drugs?

 Compared to small-molecule drugs, biologics face additional challenges. For example, the efficacy of a biologic drug may potentially be reduced through undesirable interactions of leachables with drug molecules through post-translational modification (PTM) biochemical reactions (e.g., oxidation, aggregation, clipped variants, unfolding, adducts formation, and glycosylation). Alternatively, a leachable arising from single-use systems (SUS) or components used for bioprocessing may adversely affect the manufacturing process through cellular toxicity and Chinese hamster ovary (CHO) cell death thereby reducing the productivity of the bioprocess. There are several examples where leachables have been associated with these undesirable effects on biologic manufacturing and drug efficacy, leading to major manufacturing losses and even worse, dangerous side-effects and loss of drug efficacy. 

 The evaluation of biologics for leachables presents many unique challenges. The protein itself can interfere with testing, so removal prior to analysis may be warranted. But if care is not taken, this process can unintentionally remove potential leachables, resulting in false negatives, or it may lead to contamination of the sample that may result in the generation of false positives. 

	Other ingredients in large-molecule formulations, such as polysorbate 80 and other surfactants/stabilizers, can also cause issues. These compounds often interfere with chromatographic analyses in the form of multiple large peaks that display numerous ions by mass spectrometry throughout the retention time window. These large surfactant peaks can easily mask leachables. Furthermore, proteins, surfactants/stabilizers, and other ingredients in large-molecule formulations are difficult to clean from mass spectrometers and, therefore, may carry over from one analytical run to the next if not dealt with properly. 

 The likelihood of leachables in any drug product will depend on the packaging materials, type of formulation ingredients, and conditions of use. The occurrence and impact of leachables in biologic products can present greater challenges compared to that of small synthetic molecules due to several factors. Biologics are living molecules that can be difficult to solubilize and stabilize, and quality attributes are not easily characterized compared to small molecules. The formulation ingredients for biologics often contain co-solvents or surfactants and will have more propensity to extract chemicals from packaging materials compared to typical small-molecule formulations. 

	Biologic products are complex and very sensitive to their environments. Extractables or potential leachables that may migrate into a drug product have the potential to interact, and therefore, affect the product quality, safety, or stability. In general, biologic products are formulated to solubilize, stabilize, and optimize pharmacokinetic properties consistent with the route of administration. Anything that migrates from the packaging that could interfere with this optimized environment is of concern. This includes interactions with active or excipients in a drug product formulation that lead to said quality, safety, or stability issues.

	Additionally, large molecules have greater surface areas with sites that have a propensity for interactivity based on polarity and charge. This can lead to conformational modifications and other interactions that may impact product quality.

	Primary packaging and container closure systems

 What are the key considerations when selecting primary packaging material for biologic drug products?

With every drug product and especially biologics, the most inert primary package possible must be chosen to minimize the potential for interactions to occur. Potential leachables are not the only interaction of which to be wary. Because of their reactive nature, biologic drug products can adhere to surfaces or absorb into materials. An understanding of possible interfacial interactions must be a consideration. In addition, there are other packaging considerations that must be addressed, such as container closure integrity, particle generation, and other performance concerns.

 For any drug product, it is crucial to ensure the packaging does not adulterate the drug product. Any compounds that leach from the packaging could affect the product in a variety of ways, including impacting patient safety if compounds are toxic or interfering with other analytical assays during release testing. 

	There are a number of other potential E&L risks that are unique to large molecules. Leachables may cause conformational changes in the protein or may cause the protein to aggregate. Large-molecule drug products may also chelate inorganic leachables. These types of interactions can increase the toxicity of the drug product, reduce the product’s efficacy, or affect the product’s stability. It is, therefore, important to fully evaluate the E&L risks to avoid costly delays in getting a product to market. 

What components in a container closure system can pose E&L risks to a biologic drug product?

 Typically, the container and closure components that come into direct contact with the drug product usually have the highest impact in terms of leachables observed. However, there have been many examples of leachables arising from package labels such as the inks or adhesives, as well as from secondary packaging components. These risks should, therefore, be assessed during bioprocess development.

Nearly any component in a container closure system may pose E&L risk to a biologic. Because many biologics are packaged in prefilled syringes, some of the most common components of concern are rubber stoppers. Rubber stoppers are notorious for containing nitrosamines and polynuclear aromatic hydrocarbons (PAHs), both of which are carcinogenic. Glass prefilled syringes are another common example of a component type posing a special risk to biologics. During manufacturing, tungsten pins are used to hold open the fluid path in the syringe barrel. Because manufacturing occurs at extremely high temperatures, the formation of tungsten oxides is possible. The residual tungsten oxide on the glass syringe can then leach into the final biologic drug product and cause protein aggregation or degradation. 

The most common primary packaging system for a biologic drug product is a vial system. This system is composed typically of a glass vial with an elastomeric rubber stopper and an aluminum seal with a plastic flip-off button. The other common primary package is a prefilled syringe system, which is typically a glass syringe with an elastomeric plunger and a tip cap or needle shield. Each of these components has the potential to leach substances into a drug product with contact over time. Of course, the extractables of most significant concern from glass materials are metal ions. It is well known that some biologics drugs are sensitive to various metal ions. Although these reactions are drug-product specific, these reactions are a consideration when evaluating packaging components.

	Other types of extractables are expected from elastomeric components. Elastomeric components are composed of much more than just the base polymer. Elastomer formulations typically have six to 12 added ingredients that are mixed with the base polymer under heat and pressure. This process causes chemical crosslinking to occur, which result in the formation of reaction products. These reaction products, along with residual compounds of the original raw materials, may interact with the active drug product or environment. Many of these compounds are organic; some may be inorganic, and, therefore, provide an additional source of metal ions.

	In the case of a prefilled syringe system, there is the potential for even more extractables. A glass syringe may be formed with the use of a tungsten pin. This can result in tungsten residuals that are known to interact with proteins. Another issue is that syringes typically use silicone oil as a lubricant for easier plunger movement. Silicone oil can migrate into the drug product and silicone oil droplets can act as a nucleus for particle formation/growth and protein aggregation.

	Newer packaging components are now being introduced to the industry; for example, engineered polymers are replacing glass. These polymers, such as cyclic polyolefins, are much lower in extractables and have lower surface tension characteristics that make them suitable for biologic drug products.

Why is it important to fully characterize contact materials and understand the material of construction for the container closure system and their associated E&L?

 Fully characterizing contact materials is crucial to ensure the materials chosen do not negatively affect the safety or efficacy of the drug as a result of leachables. Ideally, multiple options for container closure systems should be evaluated during the initial extractables screening. Then the packaging with the lowest risk can be selected. Establishing an extractable compound profile helps to ensure that the observed compounds are not overlooked during subsequent leachables evaluations. The constituents of large-molecule drug products often interfere with the analytical tests used to evaluate E&Ls. By establishing a material’s extractable profile, leachable analysis by mass spectrometry, using extracted ion analysis, can specifically target those compounds to evaluate their presence in the drug product. This technique effectively eliminates any matrix interferences and ensures leachables are not overlooked.

 What assessments should be performed to evaluate the potential risks of E&Ls from primary packaging that meets the biologic drug product?

 It is important to take a risk-based approach to choosing and evaluating the packaging components. It should start with supplier information on the components or system, addressing questions such as:

		What are the basic material characteristics of the components?

		Are there special needs associated with the biologic drug product application, such as the environmental conditions of storage?

	Once this information is gathered, basic evaluation by standard compendial methods is needed for compliance and allows one to begin to ‘qualify’ a component for use. But this is only the first step in proving suitability. Once compendia requirements are passed, material characterization is essential to better understand what may be extracted from the material (at levels critical to the drug product).

	The following highlights the best practice recommendation for addressing E&L for a primary package:

		Material characterization: Each individual component should be assessed to assure it has broad applicability for the application.

		Controlled extractables study: This study is a comprehensive program to understand what could be extracted from the components under a broader series of solvents, if the material characterization information is found not to be sufficient. It is crucial to perform a risk assessment to decide if this step is needed, and to determine the appropriate next step in the process based on the application. The solvents used should be aqueous-based, with considerations for organic solvents (if needed), pH, extraction conditions (such as time), extraction methods, material-to-solvent extraction ratios, etc.

		Simulation study: Depending on the drug product application, it may be appropriate to complete a simulation study, instead of going directly into a leachables study. This study is highly probable when it is especially challenging to reach the analytical evaluation threshold (AET). This may occur in a circumstance such as when evaluating a large-volume parenteral (LVP) application where there is a significant volume of drug solution. If many extractables are found from the controlled extractables study, the simulation study is a way to help identify the probable leachables to target in a formal leachables study.

		Data assessment: To determine the targets for a leachables study, it is important to evaluate the risk in the specific drug application.

		Leachables study: Method development and validation for specific leachables in the drug product should occur. Leachables testing should be conducted over drug product shelf life, at both room temperature, and accelerated conditions. The leachables should be identified based on the safety concern threshold (SCT). The SCT is the threshold dose below which a leachable would present negligible safety concerns for carcinogenic and noncarcinogenic effects. The recommended SCT for parenteral drug products, per the Product Quality Research Institute (PQRI) Extractables & Leachables Working Group for parenteral and ophthalmic drug product (PODP), is 1.5 ug/day (as described in an April 2018 workshop). 

		Special considerations for biologics include: biologic activity, efficacy, degradation, oxidation, chemical modification, immune adjuvant activity. 

 Typically, the first step is to perform an extractables profiling study on the packaging component of interest to identify the potential list of leachables in the drug formulation. The profiling study results may be used to perform a risk assessment with two goals: 

		To identify potential ‘bad actors’ from the list of extractables through predicting toxicity or performing toxicology experiments 

		To select components that have more desirable extractables profiles for the final process and eliminate components found to likely contribute to an undesirable leachable.

 How do you identify and quantify potential E&L from container closure systems?

 The first step is to expose the components of the container closure system to several model extraction solvents at exaggerated conditions of time and/or temperature. The resulting solutions are then screened by headspace and direct injection gas chromatography–mass spectrometry (GC/MS) for volatile and semi-volatile organic compounds, liquid chromatography–mass spectrometry time of flight (LC/MS–TOF) for non-volatile organic compounds, and inductively coupled plasma-mass spectrometry (ICP/MS) for elemental impurities. Additional testing methods may be used if appropriate, such as those specific for halide ions, nitrosamines, or PAHs. At Eurofins, we use the Wiley/National Institute of Standards and Technology (NIST) databases to identify compounds detected by GC/MS. For those compounds detected by LC/MS, we have a propriety database, the Eurofins Extractables Index, containing more than 1500 non-volatile organic compounds. If a compound cannot be identified via the database, additional testing may be necessary. Not only does this additional testing require advanced instrumentation (e.g., quadrupole time of flight [Q-ToF]), but it also requires the expertise of experienced and highly educated analysts.â©

 Establishing a holistic extractables profile for an article of interest is a complex and intensive process involving the use of a variety of analytical technologies. Gravimetric studies and total organic and inorganic carbon analysis are often performed to gain an understanding of the total extractable content. Fingerprinting of extracts using spectroscopic methods such as ultraviolet–visible spectroscopy (UV–VIS) and Fourier transform infrared spectroscopy (FTIR) resulting in generic information about constituent chemical classes is also common. 

	These methodologies are typically followed by more specific qualitative studies to identify volatile, semi-volatile, and non-volatile extractables using GC/MS and LC/MS techniques respectively (including high resolution accurate mass [HRAM] determination). Compounds are usually identified above the (AET) that has been determined for the material or article of interest. The AET for an article of interest depends on the target dose and number of doses expected to be stored in the container closure system or component. Analytically, the AET is used to estimate a detector response threshold using a set of reference standards carefully selected to represent the chemicals expected to be extracted. Once a list of extractable peaks above AET is identified, relative quantitation is also performed to better inform risk assessment.

	In parallel, it is also important to identify any elemental impurities that result from the extractions. This [assessment] is usually performed through either inductively coupled plasma optical emission spectrometry (ICP–OES) and inductively coupled plasma mass spectrometry (ICP–MS) methods depending on required specificity and sensitivity.

 Which analytical techniques are robust enough to identify potential E&Ls?

There is no one method that will identify all potential E&Ls. Multiple analytical techniques are needed for comprehensive assessment of extractables and leachables. For inorganic species, ICP/MS or OES are typically employed. GC/MS and LC/MS are the most common techniques for detection and identification of organic compounds. There are various LC/MS configurations for robust non-volatile organic analysis. Various additional features/techniques can improve sensitivity. One such example is ion mobility and Q-ToF to enable more precise analyses and identification of unknowns by combining ion mobility and mass-to-charge ratio.


	Vol. 31, No. 8
	August 2018
	Page: 30, 32–34

	When referring to this article, please cite it as A. Siew, “E&L Risk Assessment for Biologic Drug Products," 

Materials in contact with a drug must be fully characterized to ensure they do not negatively affect the safety and efficacy of the product.

E&L Risk Assessment for Biologic Drug Products

Q: I am a regulatory professional working for a small company with a product in the early stages of development. Can you tell me when I need to submit the extractable/leachable information for my product?

	A: There is a lot of information on what extractables and leachables are but there is little information on when this information should be submitted to the regulatory authorities. The best place to start in answering your question is to define what extractables and leachables (E&L) are and why they are considered important.

	Extractables are defined as chemical compounds that can be pulled from the primary container/closure components into the drug product. Basically, they are generated by the product and the packaging interacting over time, usually in the presence of a solvent under extreme condition of time and temperature.

	Leachables are slightly different and are defined as compounds that leach or migrate into the product from the interaction between the product and the container/closure system.

	The regulatory requirements that justify performing E&L studies are defined by 21 

) 211.94(a), which states “Drug product containers and closures shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug beyond the official or established requirements” (1). Regulation 21 

600.11(h) states, “All final containers and closures shall be made of material that will not hasten the deterioration of the product or otherwise render it less suitable for the intended use. All final containers and closures shall be clean and free of surface solids, leachable contaminants, and other materials that will hasten the deterioration of the product or otherwise render it less suitable for the intended use” (2).

	While the regulations in the United States seem to deal with extractable/leachables from the container/closure system, when deciding what to submit in your registration you should also consider your manufacturing equipment as potential sources of extractables and leachables.

	This can include, but is not limited to, filters, tubing, and/or equipment materials of construction as indicated in the European regulations.

Volume 4, Part 1, Chapter 3 addresses the extractable/leachable concept by stating, “Production equipment should not present any hazard to the products. The parts of the production equipment that come into contact with the product must not be reactive, additive, or absorptive to such an extent that it will affect the quality of the product and thus present a hazard” (3). Traditionally, E&L data were gathered and submitted in the late stages of the drug development process. Packaging suppliers were often able to provide an extractable/leachable package for their materials to the pharmaceutical manufacturer in a format that could be submitted directly to the agencies. Lately, regulatory authorities are requesting this type of information for early stage clinical trial material. This change seems to have come about during the past few years. The extractables and leachables profile in clinical trial material has become a growing concern for regulatory authorities, mostly due to advances including unique packaging materials, new and novel formulations, new drug delivery systems, new combination products being introduced, the emergence of biologics and biosimilars, and the increasing use of single-use disposables systems for manufacturing.

	Why we need to submit this information is clear, but when to submit the E&L data depends on the product type, the container and closure system being used, as well as the materials and equipment used in manufacturing. If you are developing a generic with the same active and packaging components as the brand drug, the E&L report can be submitted and be available later in the process.

	If this is a new novel product associated with clinical trials or an old active being reformulated into a new dosage form (e.g., from a tablet to an injection) then you should probably have the E&L report much earlier in the process.

	If you are updating the manufacturing process of an old product to use single-use disposable systems, you should include the E&L information as early in the filing update as possible. If you are developing a new, novel product using unique packaging components and new manufacturing advancements, this information should be evaluated early in the development of the product and be available to regulators as soon as is feasible.

	Bottom line, there is no clear rule or guidance on when you need to submit E&L data. It depends on the type of product, the packaging materials being used, and the process and materials used to manufacture the product. Performing a risk evaluation on your product by asking the above questions can help your company determine when to have the extractable/leachable information available so the approval process is not delayed, and your product can be available for patients as soon as possible.

EudraLex, The Rules Governing Medicinal Products in the European Union EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use

, Volume 4, Good Manufacturing Practice Guidelines, Part 1, Chapter 3 (European Commission, March 1, 2015).


	Vol. 31, No. 8
	August 2018
	Pages: 38, 37

	When referring to this article, please cite it as S. Schniepp, "Submitting Extractables and Leachables Data to Regulators," 

Ask the Expert

The type of product, the packaging materials being used, and the process and materials used to manufacture the product will determine when E&L data should be submitted to regulators, says Susan J. Schniepp, executive vice-president, Post-approval Pharmaceuticals and distinguished fellow at Regulatory Compliance Associates.


Submitting Extractables and Leachables Data to Regulators

Cell and gene therapies are emerging as new therapeutic modalities. Unlike conventional biological therapies produced in cells, the cells themselves are developed as medicines. Full realization of their potential requires a new paradigm where technology development and manufacturing are conducted in parallel from the earliest stages of research to the clinic. New organizational and management approaches are required as well.

	From chemistry to biology-based medicines 

	Prior to the advent of biotechnology, drug discovery was focused primarily on chemistry first to mimic plant or microorganism derived molecules and progressively to synthesize a specific inhibitor or receptor ligand. Research and product manufacturing were absolutely dissociated, and each led its own life.

	The pharmaceutical development process for small molecules dictates that of 5000–10,000 chemical compounds initially undergoing laboratory screening, approximately 2.5–5% will enter preclinical testing and 0.1% will enter clinical testing (1).

	This overall process from discovery to marketing authorization of a chemical drug can take 10–15 years. In protein and monoclonal antibody development, the manufacturing process, which may involve bacteria, insect cells, or mammalian cells, is closely linked to the final product. Gene and cell-based therapies have definitively turned a page with multi-step processes based on mammalian producer cells and a resulting gene-expressing vector or cell product. Only one sequence of interest is considered as a candidate, and all the development is focused on gene and cell delivery and manufacturing to reach the optimal clinical product.

	Rapid growth in clinical study of gene and cell therapy is increasing the worldwide need for viral manufacturing technology. Much of this capacity in vector and cell manufacturing is likely to reside in contract manufacturing organizations (CMOs).

	Developing cell-engineered products is challenging because of many aspects, including manufacturing, delivery, regulatory, and testing. The need for robust and well-characterized production methods has become increasingly important to ensure that the cell therapy will be successful not only in the initial clinical phases but also through to commercialization. In particular, viral vector manufacturing is a key step in the global cell manufacturing process. Historical challenges for gene-therapy manufacturing have included poor vector quality and a lack of scalable production systems for clinical and commercial manufacturing.

	To address these needs, laboratories and companies have developed stable or transient technologies to manufacture and purify vectors for use in human gene therapy clinical trials and future marketed products. The objective is to deploy a manufacturing process template in which the downstream concentration and purification steps do not need to be customized based on the features of the genetic sequences in the product candidate. Once the crude supernatant exhibits a minimal titer and a threshold limit for protein and DNA content, the downstream purification process can be applied.

	The aim is to develop a robust gene therapy development pipeline based on proof-of-concept data. The scalable and customizable design of a gene and cell manufacturing platform must have the capacity and flexibility to support clinical development and future commercialization of viral-based gene therapies across a broad range of programs.

	A contradictory debate has emerged between scientists who want to translate their discoveries rapidly into first-in-man studies and manufacturers who aim to industrialize the global process to increase robustness and reproducibility. In reality, both views on the situation are required. The chimeric antigen receptor (CAR)-T cell story is expected to be a first step in the cell-based therapy field. New generations of CAR-T cells, engineered dendritic cells, are in development and should enter into clinical stages in the near future.

	All these first-in-man studies must be facilitated and accelerated by manufacturing facilities able to support these gamechanging therapeutic approaches with standardized processes and expertise.

	At the same time, a global process is needed to gain efficiency to drive down costs. Autologous but also allogeneic CAR-T cells’ manufacturing price is greatly impacted by the proliferation and viability of patient T cells during the manufacturing process. A sufficient number of engineered cells is required to reach a therapeutic benefit. The experience to date shows that the cell-based therapy field must encourage research institutes, hospitals, and manufacturers to work closely and build a continuum from research to market. For example, process development aims to result in large-scale production without losing the vector and cell’s basic properties, some of which have yet to be really defined. The industry has begun to understand the proliferation specifications of an engineered CAR-T cell but what about cell biomarkers of expression, phenotype, or cell culture duration on the expected clinical result? A step-by-step process needs to be implemented to encourage or force stakeholders to define these specifications.

	Initial first-in-man study triggers rollout 

	During the early years of cell- and genetherapy development, the choice of delivery tool was more related to laboratory knowhow than to the real need of the clinical protocol. Vector design and manufacturing were restricted to experts, and once preclinical studies have had been initiated, it was difficult to turn back. The cost in development time of not considering the downstream needs up front was significant.

	One example is the case of a rare and devastating genetic disease called Junctional Epidermolysis Bullosa (JEB). The clinical protocol was known since the 2000s based on retroviral-engineered skin patches from biopsies. Questions about the vector (retroviral vs. lentiviral), the manufacturing mode (packaging cell line vs. transient transfection), or the vector pseudotype (Ampho vs. VSV-G) sharply slowed down the first-in-man studies. Other concerns such as cGMP facilities and funding were also time-consuming. The results of the first one-clinical study were reported at the European Society of Cell and Gene Therapy in 2017 by the stem-cell biologist and physician Michele De Luca of the University of Modena (2). It took more than 15 years to answer all the questions and to reach the first-in-man results.

	Since then, companies and specialized institutes have initiated multiple clinical trials based on this first treatment showing that an initial first-in-man study is fundamental to trigger such an enterprise. But during these years, skin-cell manufacturing has significantly changed with the development of embryonic stem cells, and future clinical trials will have to consider these improvements.

Delivery tools shorten development steps Delivery technology remains crucial for gene therapies as well as for cell therapies considering immune tolerance strategies using human pluripotent stem cell-derived allografts. The delivery technology is designed depending on the target cell characteristics and the clinical protocol as described in 

	For example, integrative lentiviral vectors (iLVs), which deliver DNA integrated into the host cell genome, are a leading 

delivery method for treating genetic diseases and CAR-T cancer immunotherapy. Furthermore, lentiviral vectors are really efficient for transducing primary and stem cells. In parallel, adenoassociated virus (AAV)-based vectors are used for direct 

injection especially into muscle, liver, or eyes. Both iLV and AAV technologies lead to stable and longterm gene expression and are good candidates to replace a defective gene with a functional copy, which is the very principle of gene therapy.

	The emergence of new technologies, such as gene-editing that require the coexpression of several factors, brings RNA delivery technologies into center stage. Combining transient expression with safe and efficient RNA delivery remains a challenge, particularly with new therapeutic approaches based on gene-editing. Non-integrating viral technologies or non-viral approaches (electroporation or lipid nanoparticles) are emerging at the research level and are moving toward clinical research to demonstrate their potential in terms of gene transfer and cell preservation.

	The next wave of these novel therapeutics for patients changes the regulatory and manufacturing environment. Cell manufacturing includes cell extraction from patients, cultivation, engineering, amplification, cryopreservation, and reengraftment into patients. The engineered living cell-based medicines must exhibit expected cell modification while maintaining all initial cell properties, including phenotype, viability, proliferation, and stability.

	Engineered cell therapies imply manufacturing that requires the use of highly concentrated but also highly purified vector preparations to ensure cell preservation. For example, lentiviral vectors are large and complex macromolecular assemblies of proteins, lipids, and RNA and are secreted by producer cells in a cellular culture media containing proteins and DNA contaminants. All these characteristics greatly increase the difficulty in determining which sample components are associated with the vector and which are indeed contaminants in the supernatant. Protein impurities are the most abundant contaminants in vector supernatants. They mostly arise from serum and producer cells, and the proportion of stress proteins might increase while performing a serum free cell-culture process.

	Integrating vector/cell-manufacturing processes

	Production methods are designed to preserve the vector integrity and the production batch quality. Each parameter of the production process such as the presence/absence of serum, sodium butyrate induction, or vector harvest times may have an impact on the initial crude lentiviral vector supernatant composition. Concentration and purification methods based on ultracentrifugation, tangential flow ultrafiltration, or chromatography must remove cellular debris, membrane fragments, residual DNA, and proteins that are unsuitable for transducing delicate and specialized cells. It is crucial to define a lentiviral vector composition which, by virtue of the high titer and purity, minimizes the deleterious target cell phenotypic changes that occur following transduction of target cells. Regulatory agencies should ask for more stringent data about cell-based medicine characterization and specifications that can have a strong impact on the clinical results. Furthermore, new cell sources will be considered to provide the final engineered medicines. Embryonic stem cells have entered the clinic and have provided promising results as potential treatments for macular degeneration, spinal cord injury, and type 1 diabetes. Final product manufacturing will need to integrate complementary quality controls and in-process checks to obtain the best therapeutic added value in a reproducible and standardized manner. A continuum from research to clinical phases is required to result in improved design of robust processes and technologies. Process characterization must be initiated at the small-scale research level to define parameter ranges in which the product characteristics will be maintained within desired ranges and to design a process that is robust and reproducible.

	Simultaneous technology and process development

	The cell and gene therapy field can no longer rely on a unique cell- or gene-specific technology. A therapeutic approach based either on gene addition or gene editing needs to consider specific delivery technol ogy. Cell engineering and manufacturing must be adapted to the cell source, and engineered cell immune tolerance development should require additional steps and technologies. This means that cell-based therapy must simultaneously industrialize its processes to reach clinical standards and remain open to implement a global manufacturing process with new components and technologies. This approach implies a strong relationship between discovery and development teams.

The changing regulatory and manufacturing environment is ushering in a new approach to drug development.

BioPharm International-08-01-2018

The Search for Next-Gen Expression Systems

FDA Is Not Playing Games with Biosimilars

Expectations for Residual Impurity Analysis Continue to Rise

E&L Risk Assessment for Biologic Drug Products

Submitting Extractables and Leachables Data to Regulators

A New Paradigm in Drug Development

Maintaining Cell Line Integrity

BioPharm International, August 2018 Issue (PDF)

FDA Seeks to Revive Quality Metrics Initiative