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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Biopharma seeks alternatives that meet the needs for next-gen biologic drug production.
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 Myceliophthora thermophila (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 Pseudomonas 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 Myceliophthora thermophila 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 Pichia pastoris, 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 Pichia 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 in-silico 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
When referring to this article, please cite it as C. Challener, “The Search for Next-Gen Expression Systems" BioPharm International 31 (8) 2018.