Perfusion Culture System

Perfusion Culture System, Perfusion Bioreactor, Perfusion Bioreactor design, Application

1. Perfusion Culture System By Bandhan Daripa

2. Content- Introduction Perfusion Bioreactors Perfusion Technology Advantage & Disadvantage of perfusion technology Application Conclusion References

3. Introduction Perfusion, or upstream continuous bioprocessing, has been practiced since the 1980 s towards maximizing facility utilization, expanding process flexibility and minimizing costs. Innovative technologies, sophisticated control logic systems, cell culture supplements and single-use assemblies have tremendously simplified implementation, establishing perfusion as the cornerstone of intensification and continuous processing. A perfusion culture is one in which waste medium is continuously removed from the culture and the displaced medium is replenished with fresh medium .

4. What is Perfusion Cell Culture? A perfusion cell culture process involves the constant feeding of fresh media and removal of spent media and product while retaining high numbers of viable cells. Removing spent media while keeping cells in culture can be done using alternating tangential-flow(ATF) and standard tangential-flow filtration(TFF). Another option is to retain the cells by binding them to a substrate in bioreactor. Other method include use of centrifuges. Lately, a revival of methods using acoustic waves has been seen.

5. Perfusion Bioreactors Perfusion technology for biopharmaceutical production Leading focus of R&D of any Biopharma. It involves addition of media or key media constituents at regular intervals along with retention of cells in the reactor. Many manufacturing company involved in perfusion-based systems especially for hybridoma-based monoclonal antibodies. In perfusion-based process product yield increased significantly in comparison to conventional fed-based processes. Factor VIII (ReFacto) and IgG are two of the leading products being produced commercially using perfusion technology.

6. Cont… Why perfusion technology? Its offers the ease of continuous culturing of cells without nuisance of filter clogging or low throughput. Less possibilities of waste accumulation and, hence, minimized chances of any product inhibition, especially while dealing with proteins prone to instability . The availability of key media constituents is maintained consistently by providing host cells a stable environment leading to a high cell density and higher productivity with respect to desired compound. Continuous perfusion was calculated and found to be the most productive technology giving product at the rate of 265kg/year as compared to 130kg/yr in fed-batch mode. Perfusion technology offers a lucrative mode of production especially as it beats the conventional fed-batch system in terms of productivity, efficiency, and capital investments.

7. Cont… Cell retention in Perfusion Ability to yield a high cell mass due to the presence of cell retention devices. Cell can be retained by making them grow inside bioreactor on hollow capillary fibers, flat plates, sponge-like materials, microcarrier particles, or other membranes. Also cell can be retained by use of various cell separation devices like gravity-based cell settlers, spin filters, centrifuges, cross-flow filters, alternating tangential-flow filters, vortex-flow filters, acoustic settlers, and hydrocyclones. Spin filter was one of the earliest available devices for cell retention which used a two-dimensional screen to retain the cells. Alternating tangential-flow filters(TFFs) have emerged as the most effective and practical means of high-density cell retention in a perfusion bioreactor.

8. Cont… Perfusion and bioreactors Perfusion further minimizes the losses associated with batch failure due to contamination. Even if contamination occurs earlier in the process, lesser media and other consumables would be wasted. In a recent report, a novel microfluidic cell retention device based on inertial sorting was tested positively for retention of IgG1 producing chinese hamster ovary(CHO) cell line. Parameters tested were cell retention efficiency, biocompatibility, and scalability. There was also the flexibility of configuring the device to separate different-sized cells with a specific input flow rate, this gave an added advantage of flushing out non viable cells. Perfusion microfluidic systems can also be used for growth and expression of proteins from bacterial cells.

9. Procedure for the design of perfusion bioreactors 2-step procedure for the design and development of CHO cell perfusion cultures Step 1: Definition of suitable minimum cell specific perfusion rate (CSPR) value at constant perfusion rate (P) or constant viable cell density (VCD). Step 2: Process optimization at constant cell specific perfusion rate (CSPR) but elevated values of viable cell density (VCD) and perfusion rate (P). Procedure enables high product yield and improved process performance. Product quality at steady state only shows minor variations.

10. Perfusion Culture

11. Perfusion technology Characterized by the continuous addition of fresh nutrient medium and withdrawal of an equal volume of used medium Need of perfusion Product is unstable Product concentration is low Perfusion Technologies Enhanced sedimentation Conical settlers Incline settlers Lamellar settlers Centrifugation Spin filters External Internal

12. Comparison between perfusion and fed-batch processes Perfusion Fed-batch Requires few starts Homogenous product quality throughout. Technology for cell retention is usually proprietary and it requires specialized equipment Requires constant monitoring Larger costs for waste handling and disposal Higher process contaminants/product ratio Few qualified and expert CMOs available short residence times(ideal of labile products) Multiple starts for equivalent throughput . Consistent product. It requires careful selection of harvest time. Fed-batch does not required additional and/or specialized equipment. Requires sporadic monitoring Higher costs for waste handling and disposal Lower process contaminant/product ratio Large selection of capable and licensed CMOs

13. Advantages of Perfusion Technology Better economics High cell density High productivity Longer operation duration Small fermenter size Flexibility Fast start up in process development Constant nutrient supply Better controlled culture environment Steady state operation Ease of control Better product quality

14. Disadvantages of Perfusion Technology Contamination risk Equipment failure Increased analytical costs Long validation time Potential regulatory / licensing issues

15. Application A process can be fully continuous or semi-continuous and many groups are adding continuous process technologies to key areas of manufacturing in order to improve efficiency, solve issues with facility fit, create a multiple product facility, protect sensitive proteins, etc. Another strategy for using an upstream continuous process can be for developing fast, small, and scalable manufacturing for regulatory and toxicology studies. This provides a real advantage in terms of speed if a candidate were successful in initial studies.

16. Conclusion In conclusion perfusion culture can offer several benefits that may help those in process development and manufacturing address key issues. The decision whether this technology is beneficial to specific products or manufacturing platforms is application dependent. While the benefits may be clear in some areas, there are still some questions that need to be address by companies’ looking to adopt this technology. Issues like what constitutes a batch, operation validation, in-process testing and how continuous upstream connects to the rest of the manufacturing platform needs to be carefully considered.

17. REFERENCES Michael_C._Flickinger-_Upstream_Industrial_Biotec(b-ok.org).pdf Perfusion Cell Culture :: Repligen Perfusion Medium Development for Continuous Bioprocessing of Animal Cell Cultures | American Pharmaceutical Review - The Review of American Pharmaceutical Business & Technology Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture | IntechOpen

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