Streamlining Upstream Processing: A Good Place to Start

Published on: 
BioPharm International, BioPharm International-06-01-2019, Volume 43, Issue 6
Pages: 18–20

As cost and time pressures within biopharma are on the rise, innovative expression systems may offer companies a good opportunity to streamline processes early on.

It is well-documented that biopharma companies are under increasing pressure to reduce timelines and costs associated with bringing a new biologic medicine to market. While some efforts to improve time and cost efficiencies have been made through single-use systems and novel production methods, such as continuous processing, there may be overlooked potential in innovative highly productive expression systems that could aid in the ultimate goal of bringing safe, effective biotherapies to market quicker and cheaper.

As reported by Allied Market Research, the global biopharmaceuticals market is anticipated to experience significant growth over the coming years, potentially reaching more than $500 billion by the year 2025 (1), with monoclonal antibodies (mAbs) expected to maintain dominance as the main type of product in commercial development. In terms of expression systems, mammalian-based expression systems (human-like) are used for most biologics, particularly for mAb bioprocesses, yet these are also attributed with relative high cost and low efficiency.

“Essentially, there are two key challenges currently facing the biopharma industry,” says Abhijeet Kohli, product manager at Thermo Fisher Scientific. “When it comes to traditional mAb processes, overall timelines are constantly being challenged and there are continued calls for faster commercialization. Here, the traditional processing methods the industry follows are proving to be something of a bottleneck.”

The second challenge for Kohli relates to the new types of biologics that are entering the pipeline. “The bispecific and trispecific molecules that are increasingly the focus of immuno-oncology efforts presents a further challenge for biopharma,” he notes. “These molecules are much more complex than traditional biologics and consequently have lower titers, so there is a lot of room to optimize manufacturability, reproducibility, and stability.”

Current systems: benefits and limitations

The current “work horse” in expression systems for biopharma is the Chinese hamster ovary (CHO)­ mammalian cell line, confirms Michael A. Cunningham, associate director, Upstream Manufacturing Sciences and Technology (MSAT), Life Sciences-Process Solutions, MilliporeSigma. “These expression systems most commonly rely on antibiotic or metabolic selection mechanisms to generate high-expressing cell clones.”

Mark Emalfarb, chief executive officer of Dyadic International, emphasizes that there are limitations on specific expression needs encountered with all cell lines. “The greatest disadvantage of CHO, for example, is its low natural productivity and high cost of drug development on a gram‑per‑liter basis production,” he says. “CHO also has a long production time line-41–54 days from pre-inoculum to production bioreactor and 14–21 days for fermentation process with complex expensive cell media and buffer requirements for cell viability. All this leads to an expensive cost of goods sold (COGS).”

Additionally, Emalfarb notes that with CHO cell lines there is a need for expensive virus inactivation, which must adhere to strict regulatory requirements. “Mammalian cells may harbor or become infected by viruses which could render all the previous work of no value, or even destroy the manufacturing facility’s value,” adds Terence Ryan, chief scientific officer of iBio. 

A further potential disadvantage of conventional mammalian cell lines may present itself in the field of next-generation medicines, such as bi/multi-specific antibodies as well as gene and cell therapies, clarifies Dr. Fay Saunders, head of upstream mammalian cell culture, process development, at FUJIFILM Diosynth Biotechnologies, UK site. “Mammalian cells are still limited in their ability to be able to express more complex, non-natural ‘designed’ molecules.”

Yet, CHO and other mammalian cell lines are capable of producing large, complex proteins with post-translational modifications (PTMs), which are similar to those produced in humans, Emalfarb stresses. 

In addition to mammalian/CHO cell lines, bacterial, insect, and yeast systems, which according to Dr. Nicholas Holton, R&D manager at Leaf Expression Systems, all dominate the landscape of biologics production. “The use of plants for biologics production (plant molecular farming) has been around as a nascent field for many years but has historically been held back by significant underfunding as the industry instead focused on improving the safety profiles and yields of conventional systems,” he says. “However, now that plant expression technology has finally matured and proven its commercial viability, it is increasingly being recognized as a valid commercially viable manufacturing option for diagnostic and therapeutic products.”

Ryan also notes that plants can carry out most of the post-translational modifications exhibited by mammalian cells, and any additional mammalian-specific factors necessary for maturation of a biologic can be added with additional vectors. “Plants do not naturally exactly recapitulate human glycosylation (neither do rodent cells like CHO or myeloma), but this is generally not an issue, and modified plants with more human glycosylation capabilities can serve as hosts, and mAbs produced in plants have been shown to have more potency in antibody-dependent cell-mediated cytotoxicity (ADCC) assays than those made in CHO,” he says. “Using stably-transformed plant cells (Protalix) has some of the time issues of mammalian cells due to the need to find just the right clone and coax it into performing in large bioreactors, but plant-manufactured proteins are well tolerated by humans and non-immunogenic in sustained administration.”  

“Bacterial cells, such as E. coli [Escherichia coli] cell lines, however, are unable to produce complex, mammalian-like glycosylation due to the absence of the necessary enzymatic components and the intracellular compartmentalization required,” Ryan adds. Although, as he points out, bacteria offer a cheaper alternative to mammalian cell lines. “Insect systems can also be useful,” Ryan continues, “but haven’t really broken through (except for a flu vaccine) yet, and there is a potential risk in that baculoviruses can be taken up by mammalian cells, which is little appreciated.”

A major disadvantage for yeast expression systems is the relatively low yield achieved when using these lines, explains Ronen Tchelet, Dyadic’s chief scientific officer. “Yeast expression systems have a relatively low yield in comparison to the current CHO cell lines and the production of high mannose residues within the expressed PTMs (50–200 vs three molecules in human cells, as part of either N- or O-linked glycan structures),” he adds. “This change in the glycoform’s structure may confer a short half-life and render proteins less efficacious and immunogenic in humans. C1 is head and shoulders above this cell type for the reasons noted above.”

Higher titers are always desirable within the industry, but when titers are driven primarily by transgene copy numbers, there is a possibility that genetic loci can become unstable, which can lead to titers lowering during the manufacturing process, reveals Cunningham. “Furthermore, high titer processes that are driven predominantly by maximizing biomass can make downstream processing complex, impacting product quality.”

Looking at the foreseeable future


As has been discussed earlier, there is an increasing emphasis being placed upon speed and cost within biopharma, states Ryan. “At iBio, our technology obviates the need to spend months isolating a cell clone, allowing process development to begin within a month of knowing the target gene’s sequence.”

The cost and time pressures, which Holton notes are already considerable for the industry in terms of conventional expression systems, will only propagate with the advent of more personalized medicines as well as growth in the biologics and biosimilars markets. “Mammalian cell lines are slow to develop and expensive to scale into production. In the coming decade, a move towards rapid cheap and scalable expression technologies, which are capable of producing biologically active human proteins, such as plant transient expression, will begin to attain a growing market share,” he says. “The pharmaceutical sector is extremely conservative and risk averse, so these changes in production will likely not occur quickly.”

Concurring with the speed and cost issues surrounding mammalian cell lines, particularly CHO, Emalfarb stresses that time should not be wasted by the industry thinking that CHO lines are a viable future option of choice for the industry as it moves into this next phase of more efficient, speedy, and cost saving bio-manufacturing. “CHO cells grow too slowly, they require an enormous amount of money and energy to feed with nutrients and expensive media to force the CHO cells to grow and produce relatively low levels of protein per day resulting in high capital and operational expenditure. All to get a mediocre gram per liter output. The COGS here don’t make sense compared to our C1 fungal platform for example,” he says.

For Cunningham, CHO-based mammalian cell expression systems will maintain dominance of the bioprocessing space, at least for the foreseeable future. “However, efforts will also continue to develop non-CHO expression systems, particularly to support vaccine and gene therapy applications,” he adds. “I anticipate that, given the pressures to reduce biopharma costs, there will be continued research focused on increasing speed and reducing cost to clinic in order to accelerate the bench to bedside timeline.” 

The ability to continually manufacture a product rather than through batch processing, perfusion, is an area of interest, according to Kohli. “Perfusion, however, presents a number of challenges, particularly around how developers assess quality and titers on an ongoing basis and whether key quality indicators remain intact throughout the entire process,” he notes. “Should products fall outside of quality parameters, for example, it’s vital to have measures in place that will segregate material that does not meet these criteria.”

“The processes of the future and those of today must rely on biomanufacturing techniques that are efficient, robust, and of high quality,” confirms Saunders. “It is, therefore, imperative that the expression systems and processes of the future continue to effectively isolate and identify the very best cell lines and strains.”

Additionally, Saunders emphasizes the point that despite considerable increases in titers being witnessed over recent years, there is still more work to be done in this area. “Difficult-to-express molecules are still expressed at considerably lower titers and improvements must be made in order to achieve suitable expression levels to make them commercially viable,” she says.

To be able to express novel entities, Saunders believes that there is a need to move away from traditional cell line or strain development. “Therefore,” she continues, “there will more than likely be a rational re-design of existing expression systems or efforts put into identifying novel systems.”

Streamlining starts upstream

“Streamlining the whole biologics process certainly starts with upstream processes,” states Natasha Lucki, product manager at Thermo Fisher Scientific. “Researchers want to improve and make the overall process more efficient to shorten timelines. The idea of getting to the market first will always be at the forefront of developers’ minds, especially as new categories of biologics come into the pipeline.”

According to Lucki as new technologies continue to enter the biopharma space, a focus for scientists and developers will be to take a holistic approach toward the product pipeline. “Developing processes that will facilitate not one, but the entire biologics production chain from upstream to downstream,” she adds. “For biopharma companies, increasing efficiency and enabling greater output while keeping quality in mind are very important.”

Matthew Jones, Dyadic’s chief commercial officer, stresses that despite it being known that CHO cell lines are less time and cost-effective than alternative systems, there is a lethargic resistance to change by the industry. Risk adverse industries need pace setters that positively disrupt. “We need CEOs of biopharma to engage and look at the viable options for quicker and cheaper development of new biologic entities,” he continues. “CHO cells grow slower, require expensive media to produce mediocre protein yields resulting in higher fixed and operating costs as well as expensive drug discovery processes.” 

Emalfarb goes further stating, “Modern advances in the use of synthetic biology technologies has come to cell lines. Alternatives, such as Dyadic’s C1 fungal cell line, can offer more rapid growth at a lower cost, while producing higher amounts of protein per fermenter day. Not to move away from CHO to alternative lines is ignoring the incredible scientific breakthroughs and advances that are occurring faster in biopharma than Moore’s Law did for tech.”

For Ryan, biotherapeutics are at an interesting point with many novel and cutting-edge medicines under development in the laboratory. “Indeed, finding the sweet spot among several competing priorities-speed, cost of goods, biological activity, non-immunogenicity (except vaccines), and ease of purification-is important moving forward,” he says. Therefore, other expression systems, such as iBio’s transient plant expression system, may play an important role in transitioning these new products from the lab bench to the bedside of the patient, he asserts.

“The sector is under intense and growing pressure to increase R&D efficiency, to bring new drugs to market faster, and to manage costs more effectively,” summarizes Holton. “While there is no silver bullet to address all these challenges, adopting highly efficient next-generation expression systems, such as plant-based systems can accelerate progress towards these goals.” 


1. Allied Market Research, “Global Biopharmaceuticals Market: Opportunities and Forecasts, 2018–2025,”, Report (July 2018). 

Article Details

BioPharm International
Vol. 32, No. 6
June 2019
Pages: 18–20


When referring to this article, please cite it as F. Thomas, “Streamlining Upstream Processing: A Good Place to Start,” BioPharm International 32 (6) 2019.