Solve Your "Difficult-to-Express" Protein Problem

Article Contributed by Brittni Peterson, PhD and Emily Wozniak, PhD

Have you spent time, money and resources to develop a cell line that won’t produce to the level you expect? If so, you may have a “difficult-to-express” protein, which can make you feel like this:

But before you give up on your cell line - read on.

What are “difficult-to-express” proteins?

Advances in protein discovery and engineering have led to improved biotherapeutics. These unnatural proteins are highly complex, making them “difficult-to-express” by the host cell line. Traditional manufacturing platforms are designed to produce high yields of native proteins, like some IgGs, that are easily expressed by mammalian expression systems. However, as the complexity of these proteins increases, the effectiveness of traditional manufacturing platforms is compromised. This results in low production yields and high manufacturing costs that can sink a project before it leaves the research lab.

Approved recombinant biopharmaceuticals have grown steadily each year since the first was approved in 1981. This is a reflection of an increase in the number of recombinants in the clinical pipeline as well (BioPlan Associates Annual Report, 2016).  This rise in popularity is largely due to improvements in the design of human IgG that enhance therapeutic efficacy and eliminate off-target effects.

Other examples of recombinant proteins include:

  • Humanized (chimeric) or fully human monoclonal antibodies that avoid immunogenicity when administered to humans.
  • Fusion proteins can link the IgG Fc region to one or more effector molecules (cytokines, enzymes, receptors) thereby increasing their activity.
  • Bi- or tri-specific antibodies are designed to simultaneously bind two or more different types of antigens to elicit multiple therapeutic effects.

Why are difficult-to-express proteins challenging to manufacture?

1. Reliable production of high quality complex proteins can be challenging with traditional stirred-tank bioreactors

Stirred tank bioreactors often cannot provide the optimal environment for sensitive cell lines to produce difficult-to-express proteins. Common problems include the presence of microenvironments, inconsistent nutrient availability, and waste accumulation. Inconsistencies in environmental conditions result in variable protein production and can affect their impurity profiles. Additionally, variables such as shear stress, pH, gas exchange, and nutrient availability may influence metabolomics and post-transcriptional modifications that are required to produce a consistent, high quality product.

2. High yields of complex proteins often cannot be easily obtained with traditional stirred-tank bioreactors

Traditional fed-batch bioreactors have historically been the choice manufacturing system for biotherapeutic proteins because they can cost-effectively produce large quantities of easily expressed antibodies. However, fed-batch systems often fail to produce the desired amount of complex proteins due to low titers, even after process optimization. One reason for this is because inconsistent environmental conditions in these systems negatively impact the health and longevity of the cell line, which decreases overall protein yield.

3. Modifications to traditional bioreactors still do not meet manufacturing needs of complex proteins

Solutions to circumvent the challenges posed by traditional stirred tank bioreactors have been explored. One example is perfusion tank adaptors, which are filtration systems that can be retrofitted to stirred tank bioreactors and continuously harvest cell-free product while allowing fresh media to flow through the tank. Perfusion has the advantage of higher yields per volume (as described below), improved product quality, increased facility flexibility, and decreased capital expenditures. Perfusion tank adaptors extend the lifespan of the cells in production culture by continuously providing fresh nutrients, clearing waste, and collecting product. This allows for longer production runs resulting in a greater protein yield from low-expressing cell lines. 

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Table 1. Comparison of features among AcuSyst Perfusion Bioreactors, Perfusion Tank Adaptors, and Traditional Stirred Tank Bioreactors. 

However, perfusion tank adaptors, like TTF or ATF systems, still have significant drawbacks when manufacturing difficult-to-express proteins. Research suggests that they increase the level of shear stress experienced by the cells and protein products, which can negatively impact cell viability and may impact protein quality. They are also often prone to filter fouling or clogging, negating the perfusion function and disturbing the run. In addition, continuous perfusion increases the amount of media and other raw materials required to manufacture a batch of biotherapeutic protein. This drives manufacturing costs and can have a large impact on cost of goods sold (COGs). Perfusion technology has opened the door for more efficient production runs, but there is clearly an unmet need for improved bioreactors that can maintain consistent environmental conditions to increase yield and quality of complex proteins.

Cell Culture Company’s AcuSyst perfusion bioreactors provide a cost-effective solution to maximize yield and quality of difficult-to-express proteins

Single-Use, Perfusion Technology

AcuSyst single-use bioreactors are composed of turnkey cylindrical cartridges containing porous hollow fibers, where cells grow to tissue-like densities on the outside of the fibers (extracapillary space) protected from the shear stress that is generated by media flow within in the fibers (intracapillary space).

Figure 1. C3's AcuSyst Perfusion Bioreactors are able to improve product yields and reduce costs for difficult-to-express proteins. 

Figure 1. C3's AcuSyst Perfusion Bioreactors are able to improve product yields and reduce costs for difficult-to-express proteins. 

AcuSyst perfusion systems are designed to consistently deliver nutrients and remove waste, without the excessive media usage required by perfusion tank attachments like TTF or ATF systems. As described above, this extends cell line lifespan, resulting in increased yields of quality protein. To see Case Study Data, explore our website. 

Full Automation and Process Monitoring

AcuSyst bioreactors are fully automated and continuously monitor environmental conditions over the entire manufacturing process to ensure product consistency.

Healthy Cell Lines

By providing the optimal environment, AcuSyst bioreactors enable longer production runs. Typical runs with AcySyst systems may last 60–120 days, which is 4-8 times the length of healthy cell cultures in fed-batch tanks, where cultures typically decline around 14–21 days. See the data and read more here. 

Continuous Harvest

AcuSyst bioreactors are designed to continuously harvest concentrated, cell-free product over the entire duration of the culture period. Several of C3’s case studies have shown improved yields of 50-1000 times higher when manufacturing difficult-to-express proteins. To read more about these case studies, visit our website (link).


While traditional stirred tank bioreactors may not be cost-effective for manufacturing complex biotherapeutics, AcuSyst systems provide a solution. See our recent article in GEN for more about cost savings associated with AcuSyst bioreactors.

For more details on the AcuSyst bioreactors, their manufacturing benefits and applications, see this summary on our recent presentation at 2016 Biotech Week in Boston, this article in Cell Culture Dish and visit our website.

Contact us here to start a conversation with us about using AcuSyst bioreactors for your protein production needs.