Econ om ical GM P Pr odu ct ion of Dif f icu lt -t o-expr ess Pr ot ein s

Transcription

1 Econ om ical GM P Pr odu ct ion of Dif f icu lt -t o-expr ess Pr ot ein s

2 Table of Con t en t s 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)

3 Dif f icu lt -t o-expr ess Pr ot ein s: Fed-Bat ch is Not t h e An sw er Advances in protein engineering allow for more effective biotherapeutics, diagnostics, and protein reagents. Changing the design of native protein motifs has led to improvements in specificity, effectiveness, 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 effectiveness of traditional manufacturing platforms is compromised. Fed-batch results in low production yields and high manufacturing costs that can sink a project before it reaches commercialization. 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 sensitivity to environmental conditions, protein auto-inhibition, or the necessity of specific post-translational modifications. Any of these problems can add significant cost to clinical trial 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 accumulation are common problems caused by fed-batch manufacturing. This may result in variable protein quality, high presence of impurities, or low protein yields. Additionally, environmental conditions 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. A Choosing the right production platform and using an experienced, knowledgeable, and flexible team of biomanufacturing experts can save both time and money. Traditional cell culture methods and fed-batch bioreactors often cannot provide the optimal environment for sensitive cell lines to produce difficult-to-express proteins. Culture Days B Perfusion-based bioreactors with automated process monitoring capabilities are one method to achieve reliable production 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 production in AcuSyst bioreactors remains stable during these long runs, granting the flexibility to extend runs to get enough protein from a single batch for low-expressing cell lines without significantly increasing costs (Figure 1C). These results support other studies that demonstrate perfusion systems can maintain long-term cell health and protein production. Consistent metabolic parameters support reliable, high yield, and high-quality production of difficult-to-express proteins. In turn, this can only help to increase the clinical and commercial successes of these difficult-to-express proteins. Culture Days C Another consideration for managing costs, timelines, and risk with difficult-to-express proteins is the flexibility and experience of the manufacturer. When choosing a Contract Manufacturing Organization (CMO) it is critical 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. Culture Week 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). 3

4 Acu Syst Per f u sion Bior eact or s Solve Dif f icu lt -t o-expr ess Pr ot ein Ch allen ges To overcome low-expressing cell line challenges, reconsider the manufacturing platform. Typical fed-batch processes are optimized 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 days before there is a sharp drop in cell viability and protein titer. 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 time period. For difficult-to-express proteins, this could mean insufficient yield or unacceptable product quality. Perfusion-based bioreactors offer a solution. Perfusion systems provide consistent environmental conditions by continuously circulating fresh nutrients, removing waste, and collecting product. These advantages allow for longer production runs and greater protein yields from low-expressing cell lines. Traditional stirred 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, negating the perfusion function, disturbing the run, and potentially causing product inconsistencies due to mid-run disruption. Alternatively, 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 continuously harvested into a refrigerator to preserve protein quality. By providing an optimal environment, AcuSyst bioreactors were designed to enable longer production runs without the risk of filter fouling or clogging. Typical runs with AcuSyst systems may last days, which is 4-8 times the length of healthy cell cultures in fed-batch tanks, where cultures typically decline around days. Several of C3?s case studies have shown improved yields of 5-20X higher when manufacturing difficult-to-express proteins (Figure 3). 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. 4

5 Th in k Ou t side t h e Tan k Both CDMOs and companies with in-house capabilities are currently experiencing a capacity crunch. The biggest casualties 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 times. C3 took a different approach. To best meet its customers?needs, C3 designed its upstream manufacturing facilities to be flexible, thus reducing overhead and timelines. By utilizing this strategy, C3 gets customers in the door faster and more cost-effectively 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-optimal 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 facilities booked out 1-2 years in advance, and many facilities are often hit with unpredictable delays in the manufacturing process. Contamination, technical challenges, and changes in production methods can cause significant delays. Manufacturers can take measures to reduce these delays by designing their facilities to be flexible. Appropriate choices during facility set up, clean room design, and instrument choice may allow for later changes to production 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 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 single-use platforms are known to reduce the risk of contamination between batches, these systems also reduce the time in between manufacturing runs and can save on costs. Clean-in-place systems needed for operating large stainless steel tanks can be costly in capital expenditures andin valuabletime. The biomanufacturing industry is rapidly shifting 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 outlet for installation. These systems have automated temperature control and built-in product refrigeration so that they can operate autonomously without excess equipment. AcuSyst Bioreactors also eliminate the capital- and space-intensive seed train, saving both cost and time. They are ideal for flexible, multi-product facilities designed to be readily adaptable to keep biomanufacturing schedules on track. 5

6 Case St u dies: GM P M an u f act u r in g w it h Acu Syst Bior eact or s While fed-batch systems have historically dominated the marketplace, biopharmaceutical and diagnostics companies have been manufacturing protein using hollow fiber bioreactors for over 25 years. These organizations report consistency in batch yields and cost savings. While many organizations initially 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 organizations continue 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 initial 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 production inan Xcellerator(6-20 cartridges, equivalent of a 1,000 L to 2,000 L tank) without additional engineering. This saved cost and accelerated their timeline to market. Company 2 - FDA-approved Biologic: 8 batches Company 2 is a biopharmaceutical company with an FDA-approved biologic aimed at a small patient population. Because its target patient population was small, the company required only 5-10g annually, rather than the kilograms optimally produced by stirred tank bioreactors. Company 2 found a cost-effective solution to their small-batch needs by using perfusion hollow fiber bioreactors to manufacture its biologic. Company 3 - Early-stage clinical trial Biologic: In ProcessDevelopment Company 3 achieved pre-clinical success with an immunotherapy targeting pancreatic cancer. This organization chose C3 because of their aggressive timeline and their need for high levels of expertise and quality. The immunotherapy will be produced using the AcuSyst Xcellerator under GMP conditions. 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 diagnostic under GMP conditions for over 15 years. The Xcellerator (2,000 L tank equivalent) production 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 ProcessDevelopment Company 5 has an early-stage companion diagnostic 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 combination of perfusion and continuous 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 publications (Sargent, 2012; Jorjorian and Kenyon, 2017; Pollock et al., 2013; Wozniak and Biesecker, 2017; Zhang et al., 2015) showing significant cost and time savings associated with perfusion bioprocessing. These studies commonly note the reduced capital expenditures, reduced operational overhead, advantages in facility flexibility, and higher protein yields. In the case of the hollow fiber bioreactor, additional cost savings are found in raw materials and in reduced downstream concentration steps compared to other perfusion options (Waniger et al., 2017; Vermasvouri and Hurme, 2011). It is important to note that these publications, 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. 6

7 Fin d ou t m or e abou t h ow w e can m an u f act u r e dif f icu lt -t o-expr ess pr ot ein s w it h ou t br eak in g t h e ban k VISIT C3 WEBSITE 7 Produced by Generis Group generisgp.com

8 Ref er en ces Jorjorian P, Kenyon D, How to Set Up a Perfusion Process for Higher Productivity and Quality, -process-higher-productivity-quality/. Pollock J, Ho SV, Farid SS, Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty. Biotechnol Bioeng, 110(1): Sargent B, Employing Perfusion Bioreactors to Improve Antibody Production in CHO Cells, Vermasvuori R, Hurme M, Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes. Biotechnol. Prog., 27(6): Waniger S, Wozniak E, Biesecker K, Difficult-to-Express Proteins: Resolving Bioprocessing Challenges with a Scalable Perfusion Bioreactor, Wozniak E, Biesecker K, Perfusion Solutions for Therapeutics Development: Fail Fast, Fail Cheap, GEN. Zhang Y, Stobbe P, Silvander C, Chotteau V, Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor. Journal of Biotechnology, 8