Econom ical GMP Product ion of

Dif f icult -t o-Express Prot eins

Sect ion 1: Difficult-to-Express Proteins: Fed Batch is Not the Answer (page 3)

Sect ion 2: AcuSyst Perfusion Bioreactors Solve Difficult-to-Express Protein Challenges (page 6)

Sect ion 3: Think Outside the Tank (page 7)

Sect ion 4: Case Studies: GMP Manufacturing with AcuSyst Bioreactors (page 8)

Table of Cont ent s

Dif f icult -t o-Express Prot eins: Fed-Bat ch is Not t he Answer

Advances in protein engineering allow for more effect ive biotherapeutics, diagnost ics, and protein reagents. Changing the design of nat ive protein motifs has led to improvements in specificity, effect iveness, and ease of development for applied proteins. However, these unnatural proteins are often highly complex, making them ?difficult-to-express? by the host cell line. As the complexity of these proteins increases, the effect iveness of tradit ional manufacturing platforms is compromised. Fed-batch results in low product ion yields and high manufacturing costs that can sink a project before it reaches commercializat ion.

Difficult-to-express proteins are classified as such when it is challenging to generate a high enough batch yield to meet the company?s needs. There are a number of reasons why a cell line may have a low yield, including protein or cell sensit ivity to environmental condit ions, protein auto-inhibit ion, or the necessity of specific post-translat ional modificat ions. Any of these problems can add significant cost to clinical t rial manufacturing in the form of manufacturing delays, extensive troubleshooting during process development, or high manufacturing costs due to low yields.

The presence of microenvironments, inconsistent nutrient availability, and waste accumulat ion are common problems caused by fed-batch manufacturing. This may result in variable protein quality, high presence of impurit ies, or low protein yields. Addit ionally, environmental condit ions such as shear stress, pH, gas exchange, and nutrient availability may influence metabolomics and post-transcript ional modificat ions that are required to produce a consistent, high-quality product.

Choosing the right product ion platform and using an experienced, knowledgeable, and flexible team of biomanufacturing experts can save both t ime and money. Tradit ional cell culture methods and fed-batch bioreactors often cannot provide the opt imal environment for sensit ive cell lines to produce difficult-to-express proteins.

Perfusion-based bioreactors with automated process monitoring capabilit ies are one method to achieve reliable product ion of a high-quality product. AcuSyst bioreactors are an example of such a system. AcuSyst bioreactors are able to maintain stable pH, glucose and lactate levels for long runs, even up to 90 days (Figure 1A and 1B). Similarly, protein product ion in AcuSyst bioreactors remains stable during these long runs, grant ing the flexibility to extend runs to get enough protein from a single batch for low-expressing cell lines without significant ly increasing costs (Figure 1C). These results support other studies that demonstrate perfusion systems can maintain long-term cell health and protein product ion. Consistent metabolic parameters support reliable, high yield, and high-quality product ion of difficult-to-express proteins. In turn, this can only help to increase the clinical and commercial successes of these difficult-to-express proteins.

Another considerat ion for managing costs, t imelines, and risk with difficult-to-express proteins is the flexibility and experience of the manufacturer. When choosing a Contract Manufacturing Organizat ion (CMO) it is crit ical to consider their experience with challenging molecules. A CMO?s ability to ?think outside the tank? should be highly rated for products that are difficult-to-express, small batch, or have quality concerns.

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Figure 1. Consistent metabolic parameters indicate long-term cell health and antibody production in AcuSyst Bioreactors. (A) Two IgG-producing cell lines were run in AcuSyst Bioreactor culture for 85 and 70 days (Cell Line 1 and 2, respectively). Both maintained consistent glucose/lactate levels throughout the culture. (B) pH remained stable in both cultures. (C) Antibody production in two AcuSyst Bioreactor cultures for up to 15 weeks (not the same cultures as A and B).

A

B

C

Culture Days

Culture Days

Culture Week

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To overcome low-expressing cell line challenges, reconsider the manufacturing platform. Typical fed-batch processes are opt imized for high-expressing cell lines (many kg?s of product) at the commercial scale. The cell culture environment in this platform can maintain cell health for about 14-21 days before there is a sharp drop in cell viability and protein t iter. For high-expressing, naturally occurring proteins (e.g. human IgGs), this is not a problem as sufficient yields of high-quality product can be obtained in this t ime period. For difficult-to-express proteins, this could mean insufficient yield or unacceptable product quality.

Perfusion-based bioreactors offer a solut ion. Perfusion systems provide consistent environmental condit ions by cont inuously circulat ing fresh nutrients, removing waste, and collect ing product. These advantages allow for longer product ion runs and greater protein yields from low-expressing cell lines.

Tradit ional st irred tank bioreactors can be modified to be perfusion-based through perfusion tank adapters, like TTF or ATF systems. Tank adapters use perfusion to solve some difficult-to-express problems, but they have significant drawbacks. ATF perfusion adapters use large volumes of raw materials and are prone to filter fouling or clogging, negat ing the perfusion funct ion, disturbing the run, and potent ially causing product inconsistencies due to mid-run disrupt ion.

Alternat ively, AcuSyst bioreactors can improve yields of difficult-to-express proteins without increasing shear stress or increasing costs. These single-use bioreactors grow adherent and suspension cells in the extracapillary (EC) space of the hollow fibers and are protected from the shear stress generated by media flow through the intracapillary (IC) space (Figure 2). Product is retained with the cells in the EC space and cont inuously harvested into a refrigerator to preserve protein quality. By providing an opt imal environment, AcuSyst bioreactors were designed to enable longer product ion runs without the risk of filter fouling or clogging.

Typical runs with AcuSyst systems may last 60-120 days, which is 4-8 t imes the length of healthy cell cultures in fed-batch tanks, where cultures typically decline around 14-21 days. Several of C3?s case studies have shown improved yields of 5-20X higher when manufacturing difficult-to-express proteins (Figure 3).

AcuSyst Per fusion Bioreact ors Solve Dif f icult -t o-Express Prot ein Challenges

Figure 2. Schematic of an AcuSyst Bioreactor cartridge.

Figure 3. AcuSyst Bioreactors boost yields of difficult-to-express cell lines. Four cell lines that struggled to achieve sufficient production volumes in fed-batch culture were grown in AcuSyst Bioreactors. Each saw a boosted yield of ~5-fold and production of the GM-CSF fusion protein was boosted 20-fold.

Think Out side t he Tank

Both CDMOs and companies with in-house capabilit ies are current ly experiencing a capacity crunch. The biggest casualt ies are the 1g to 1kg range GMP manufacturing projects that take up fewer resources but use the same expensive overhead as projects that are 100s of kgs per year. CDMOs either hike up prices on small scale projects to offset the facility usage cost, or they require 9-18 month wait t imes.

C3 took a different approach. To best meet its customers? needs, C3 designed its upstream manufacturing facilit ies to be flexible, thus reducing overhead and t imelines. By ut ilizing this strategy, C3 gets customers in the door faster and more cost-effect ively than other CDMOs that use massive, fixed equipment like fed-batch tanks.

To accomplish this, C3 designed upstream biomanufacturing platforms that meet three main criteria: automated, small-footprint, and single-use.

Automated

Bioreactors with advanced automation reduce the risk that the environment within the bioreactor is sub-opt imal or variable, and thereby ensures batch-to-batch product consistency. Manufacturers should consider bioreactors with in-process monitoring and control of the cell culture environment, including pH, glucose, and lactate levels.

Small Footprint

Biomanufacturing batches are especially challenging to plan and schedule. Manufacturers often have facilit ies booked out 1-2 years in advance, and many facilit ies are often hit with unpredictable delays in the manufacturing process. Contaminat ion, technical challenges, and changes in product ion methods can cause significant delays. Manufacturers can take measures to reduce these delays by designing their facilit ies to be flexible. Appropriate choices during facility set up, clean room design, and instrument choice may allow for later changes to product ion spaces in response to changes in plans. Choosing instruments that are readily interchangeable, occupy a small footprint, and are standalone without the need for complimentary equipment (like a large seed train system) can ensure that a facility is ready for anything.

Single-Use

While single-use platforms are known to reduce the risk of contaminat ion between batches, these systems also reduce the t ime in between manufacturing runs and can save on costs. Clean-in-place systems needed for operat ing large stainless steel tanks can be cost ly in capital expenditures andin valuablet ime. The biomanufacturing industry is rapidly shift ing towards single-use systems in both upstream and downstream instruments in order to take advantage of these benefits. AcuSyst single-use bioreactors occupy a small footprint. The smallest fit on a benchtop and the largest (2,000 L equivalent) is about the size of a refrigerator and only requires an out let for installat ion. These systems have automated temperature control and built -in product refrigerat ion so that they can operate autonomously without excess equipment. AcuSyst Bioreactors also eliminate the capital- and space-intensive seed train, saving both cost and t ime. They are ideal for flexible, mult i-product facilit ies designed to be readily adaptable to keep biomanufacturing schedules on track.

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The AcuSyst Xcellerator is a single-use bioreactor equivalent to a 2,000L stirred tank, but it can fit through standard doorways and only requires a wall outlet to function.

While fed-batch systems have historically dominated the marketplace, biopharmaceutical and diagnost ics companies have been manufacturing protein using hollow fiber bioreactors for over 25 years. These organizat ions report consistency in batch yields and cost savings. While many organizat ions init ially chose the AcuSyst perfusion platform for cost savings in small batches, they often stay with the technology because of the reproducibility of protein quality and yield, especially during scale-up or scale-down efforts. Below are a few examples where organizat ions cont inue to use perfusion hollow fiber bioreactors for small-scale GMP manufacturing:

Company 1 - GMP in In Vitro Diagnostic: 4 batches

Company 1 outsourced manufacturing to C3 and benefited from the scalability of AcuSyst perfusion bioreactors. These bioreactors are linearly scalable, which means scale-up only involved adding cartridges of the same size in parallel. C3 performed init ial process development in benchtop AcuSyst AutovaxIDs, which have one cartridge and can produce a quantity of protein equivalent to a 100 L tank. Company 1 was able to scale up for commercial product ion inan Xcellerator(6-20 cartridges, equivalent of a 1,000 L to 2,000 L tank) without addit ional engineering. This saved cost and accelerated their t imeline to market.

Company 2 - FDA-approved Biologic: 8 batches

Company 2 is a biopharmaceutical company with an FDA-approved biologic aimed at a small pat ient populat ion. Because its target pat ient populat ion was small, the company required only 5-10g annually, rather than the kilograms opt imally produced by st irred tank bioreactors. Company 2 found a cost-effect ive solut ion to their small-batch needs by using perfusion hollow fiber bioreactors to manufacture its biologic.

Company 3 - Early-stage clinical trial Biologic: In Process Development

Company 3 achieved pre-clinical success with an immunotherapy target ing pancreat ic cancer. This organizat ion chose C3 because of their aggressive t imeline and their need for high levels of expert ise and quality. The immunotherapy will be produced using the AcuSyst Xcellerator under GMP condit ions.

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Company 4 - GMP In Vitro Diagnostic: 11 batches

Company 4 outsourced manufacturing to C3, where AcuSyst Bioreactors have been used to manufacture it sin vitro diagnost ic under GMP condit ions for over 15 years. The Xcellerator (2,000 L tank equivalent) product ion runs have never failed a batch, and the yields for each batch are consistent and predictable within +/- 10%.

Company 5 ? Early-stage clinical trial Biologic: In Process Development

Company 5 has an early-stage companion diagnost ic that could not be manufactured in batch or fed-batch systems due to build up of waste products that inhibited expression of their target molecule and compromised its quality. The combinat ion of perfusion and cont inuous harvest in the AcuSyst Bioreactor was beneficial and necessary for this company?s product to be produced at the highest yield and quality.

While these examples represent a few of the customers that have used C3?s AcuSyst Bioreactors for GMP manufacturing, more are turning to perfusion. There have been several publicat ions (Sargent, 2012; Jorjorian and Kenyon, 2017; Pollock et al., 2013; Wozniak and Biesecker, 2017; Zhang et al., 2015) showing significant cost and t ime savings associated with perfusion bioprocessing. These studies commonly note the reduced capital expenditures, reduced operat ional overhead, advantages in facility flexibility, and higher protein yields. In the case of the hollow fiber bioreactor, addit ional cost savings are found in raw materials and in reduced downstream concentrat ion steps compared to other perfusion opt ions (Waniger et al., 2017; Vermasvouri and Hurme, 2011). It is important to note that these publicat ions, and our experience over the past few decades, confirm that these advantages don?t come at the cost of quality. In fact, most studies find greater batch-to-batch consistency in product quality when using perfusion.

Case St udies: GMP Manufact ur ing w it h AcuSyst Bioreact ors

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References

Jorjorian P, Kenyon D, 2017. How to Set Up a Perfusion Process for Higher Product ivity and Quality, ht tp://www.bioprocessint l.com/upstream-processing/perfusion-cell-culture/set-perfusion-process-higher-product ivity-quality/.

Pollock J, Ho SV, Farid SS, 2013. Fed-batch and perfusion culture processes: economic, environmental, and operat ional feasibility under uncertainty. Biotechnol Bioeng, 110(1):206-19.

Sargent B, 2012. Employing Perfusion Bioreactors to Improve Antibody Product ion in CHO Cells, http://cellculturedish.com/2012/04/employing-perfusion-bioreactors-to-improve-ant ibody-product ion-in-cho-cells/.

Vermasvuori R, Hurme M, 2011. Economic comparison of diagnost ic ant ibody product ion in perfusion st irred tank and in hollow fiber bioreactor processes. Biotechnol. Prog., 27(6):1588-98.

Waniger S, Wozniak E, Biesecker K, 2017. Difficult-to-Express Proteins: Resolving Bioprocessing Challenges with a Scalable Perfusion Bioreactor, http://www.bioprocessint l.com/manufacturing/cont inuous-bioprocessing/acusyst-scalable-perfusion-bioreactor-difficult-express-proteins/.

Wozniak E, Biesecker K, 2017. Perfusion Solut ions for Therapeutics Development: Fail Fast, Fail Cheap, GEN.https://www.genengnews.com/gen-art icles/perfusion-solut ions-for-therapeutic-development/5984

Zhang Y, Stobbe P, Silvander C, Chotteau V, 2015. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor. Journal of Biotechnology,

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