Mass Production of Antithrombin Module. Generate Ideas: In class have the students write journal entries to answer the following questions:

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1 Mass Production of Antithrombin Module Challenge: The human plasma protein antithrombin is an anticoagulant that plays a key role in controlling clot formation. It is used as a therapeutic protein to prevent blood clots in patients who lack the natural anticoagulant protein. How do you mass produce this protein? Generate Ideas: In class have the students write journal entries to answer the following questions: 1. What are your initial ideas about how this question can be answered? 2. What background knowledge is needed? 3. What do you already know about mass producing recombinant proteins in microbes or higher organisms? 4. What factors are involved with scale up for producing therapeutic recombinant proteins? 5. What downstream considerations must be analyzed for designing an appropriate recombinant protein manufacturing system? Multiple Perspectives: In the whole-class setting, have the students share ideas from their journals. Record their ideas on the board. Break students up into groups and have each group review the following quotes: Proteins can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants or insects, or via transgenic plants and animals. Protein quality, functionality, production speed and yield are the most important factors to consider when choosing the right expression system for recombinant protein production. Demain, AL and Vaishnav, P., Production of recombinant proteins by microbes and higher organisms, Biotechnology Advances 27 (2009) Because of the high prices that drugs can command, medicine not food might provide the earliest payoff from genetically engineered livestock. companies are at work on medicines that would be extracted from a transgenic animal s milk or blood, saying the approach might be less expensive than other ways of making protein drugs. Pollack, A., Initial Benefit From Genetic Engineering Likely to Be Medicine, The New York Times, July 30, Have the groups formulate some more ideas based on this added information and add these ideas, along with the original ideas, to the board. Work with the class to set up main categories of necessary knowledge. If possible, have these categories focus on: 1. Molecular biology of different expression systems 2. Product quality and safety (since it is a recombinant human therapeutic protein need to ensure proper post-translational modifications) 3. Levels of expression and yields (scale up)

2 4. Manufacturability (production days/year and reactor capacity required) 5. Capital expenditures and production costs 6. Downstream processing (harvesting of crude protein, product recovery, purification) 7. Selection of ideal organism Research and Revise: Assign the following reading material associated with the generate ideas questions. These readings are intended help the students realize how molecular biology, expression systems, limitations, trade-offs, safety, yields, manufacturability, expenditures, costs, and downstream processing are all coupled together for properly selecting an ideal organism for the mass production of a human therapeutic protein. 1. Chapter 14: Utilizing Genetically Engineered Organisms. Bioprocess Engineering: Basic Concepts, 2 nd Ed., by Michael L. Shuler and Fikret Kargi, Prentice Hall, ISBN: ; Demain, AL and Vaishnav, P., Production of recombinant proteins by microbes and higher organisms, Biotechnology Advances 27 (2009) Dimond, PF., Transgenic Technology in the Production of Therapeutic Proteins, Innovations in Pharmaceutical Technologies (2000) The students work through 6 main topics associated with the 7 categories outlined in Multiple Perspectives: 1. What is known about the molecular biology of different expression systems and organisms? This material is covered in course prerequisite material and from previous lectures in the class. This is a good time to review some of the salient features of expression systems in different organisms. This discussion should highlight the significant differences between the use of prokaryotic and eukaryotic organisms. This is also a good time to apply the concepts of expressing a human protein in an appropriate organism. Previous lectures covered eukaryotic DNA processing and post-translational modifications of the expressed protein. At this point, ensure the students are expecting to use a eukaryotic organism and system for this type of protein. 2. What concepts do we need to apply for choosing an appropriate organism to ensure product quality and safety? Discuss the absolute needs of producing a safe and effective human therapeutic protein. Ensure the students clearly understand that this challenge asks for the mass production of a human therapeutic protein and that the appropriate organism would have to be at the very least eukaryotic, but better if the choice was mammalian. Help the students realize that product quality and safety can be attained from mammalian organisms or cell cultures since the proteins are often made in a properly folded and glycosylated form. This provides the best option for closely mimicking a human protein. 3. What type of organism would provide high levels of expression and yields at scaled up levels? Discuss options for expressing proteins in different organisms. Help the students understand the implications of choosing an appropriate system. This upstream system will

3 drive the manufacturing and downstream processes as well as influence economic decisions. At this point, the students may have already chosen or have a good idea of what they will choose for an organism. This part of the module will help them research the best way to implement their chosen plan in terms of expression levels and yield. 4. What is the manufacturability of the product in the chosen organism (production days/year and reactor capacity required)? Although many ways exist to produce a recombinant human therapeutic protein, ensure the students realize and understand the tradeoffs in manufacturability. Explain how similar organisms or systems may produce the same quality of protein, however each has production limitations in terms of batch sizes, production days per year, batch turnaround, and overall throughput. Continuously instill the thought process for designing a system to mass produce a recombinant therapeutic protein. 5. What capital expenditures and production costs are associated with different organisms and expression systems? Help the students understand the overall process in terms of capital equipment and the different production costs for typical commercial scale biologics production. Compare the differences in production economics for these systems and illustrate the tradeoffs between product quality, expression levels, yields, manufacturability, and downstream processing. 6. How does a chosen organism and the production process design affect downstream processing? Even though this is predominantly an upstream bioprocess engineering systems course, it is critical for the students to understand the implications of the upstream process design on the downstream process. All of the previous questions and design designs affect how the biologic product will be harvested, processed, and purified. Discuss how closely coupled and integrated the upstream and downstream processes must be to successfully manufacture a safe, effective, and economical human therapeutic protein. Test Your Mettle: Since this module is based on a challenge question that is essentially answered by selection of an appropriate organism for producing a therapeutic biologic, the students should be asked to perform their research, select an appropriate organism, and be able to support and defend their choice based on the 6 main topics the Research and Revise segment. In a whole class setting: 1. Discuss the reading assignments and focus the discussion to address the main concepts listed in the multiple perspectives component. 2. Ensure students are on the right track in terms of choosing the ideal organism by clarifying any misconceptions and answering any lingering questions the student may have. 3. Have the students work individually in class and have them choose their organism. Give them some time to jot down some notes on their rationales based on the main issues associated with this module.

4 4. Have the students briefly present to the class their organism and/or process and to defend their selection. 5. Ask the students to critique each informal presentation based on the selection and the defense. Provide support by asking questions to force the students to critically think about their selections and process designs. Clarify any misconceptions and answer any questions the students themselves cannot answer as a group. Go Public: You ve been hired as a consultant by Vandalay Biopharmaceuticals to provide information and justification for the selection of the ideal organism to mass produce recombinant human antithrombin protein for therapeutic use. You ve been asked to create an informative and persuasive PowerPoint slideshow to report and defend your selection. This slide show should be designed as stand-alone media that doesn t require a presenter. In other words, you are asked to produce a slide show that contains sufficient information that knowledgeable individuals at Vandalay Biopharmaceuticals can clearly follow and understand your rationale. (Assume your audience understands bioprocessing technologies.) See attached page

5 Mass Production of Antithrombin Module Go Public (100 pts.) You ve been hired as a consultant by Vandalay Biopharmaceuticals to provide information and justification for the selection of the ideal organism to mass produce recombinant human antithrombin protein for therapeutic use. You ve been asked to create an informative and persuasive PowerPoint slideshow to report and defend your selection. This slide show should be designed as stand-alone media that doesn t require a presenter. In other words, you are asked to produce a slide show that contains sufficient information that knowledgeable individuals at Vandalay Biopharmaceuticals can clearly follow and understand your rationale. (Assume your audience understands bioprocessing technologies.) Note: Quality slide shows and presentations use as few words as possible. Nobody wants to see full paragraphs on a slide. The most effective slides shows use bullet type statements (not necessarily complete sentences) that concisely and precisely convey the information. Your job is to persuade Vandalay Biopharmaceutical officials that your choice for an organism is ideal. You can effectively make your argument using quick facts, bulleted style statements, statistics, and other means you feel support your argument. Visually appealing slides are also helpful, but there is a fine line between unnecessary gaudiness and a pleasant presentation. Don t feel that you have to be a graphic artist with fancy backgrounds and flashy borders. Keep it simple, informative, and persuasive. To successfully convey and defend your choice for an ideal organism to Vandalay officials, your slide show must include the following components: 1. Your selected organism 2. Molecular biology of different expression systems 3. Product quality and safety (since it is a recombinant human therapeutic protein, need to ensure proper post-translational modifications) 4. Levels of expression and yields (scale up) 5. Manufacturability (production days/year and reactor capacity required) 6. Capital expenditures and production costs 7. Downstream processing (harvesting of crude protein, product recovery, purification) 8. Conclusions of your ideal organism selection 9. Citations of references (last slide) Grading Guidelines: Appropriate technical content (from list above) Clarity and Persuasiveness PowerPoint quality (Clear and professional visual aids) Readability 70 pts. 10 pts. 10 pts. 10 pts. Total: 100 pts.

6 Below is the suggested time line for the Mass Production of Antithrombin Legacy Cycle. After using it as an overview to plan out lessons, you can also make a copy to mark up and use as a checklist to keep track of what things your class has completed. This can be especially useful if you have multiple classes working through the challenge, as multiple classes are seldom at the same place at the same time in a series of lesson. page(s) LECTURE DAY ONE Introduce the Challenge Question. Students independently work in their journals to answer the Generate Ideas questions. In the whole-class setting, have students share ideas from their journals. Record their ideas on the board. Break students up into groups and have each group review the two quotes provided in the Generate Ideas segment. Have the groups formulate some more ideas based on this added information and add these ideas, along with the original ideas, to the board. Work with the class to set up main categories of necessary knowledge. Work with the class to set up the 7 main categories of necessary knowledge as listed in the Multiple Perspectives. LECTURE DAY TWO Start off the lecture by reviewing the overall goal of the module. Repeat the challenge question and list the 7 categories developed in the Multiple Perspectives: 1) Molecular biology of different expression systems; 2) Product quality and safety (since it is a recombinant human therapeutic protein need to ensure proper post-translational modifications); 3) Levels of expression and yields (scale up); 4) Manufacturability (production days/year and reactor capacity required); 5) Capital expenditures and production costs; 6) Downstream processing (harvesting of crude protein, product recovery, purification); and 7) Selection of ideal organism After the review, start the discussion by asking the students their thoughts on the level of organism (prokaryotic or eukaryotic) that should be considered to solve this challenge. Ensure the students understand the end product is a human protein and must be safe and effective for human therapeutic use. Review essential prerequisite material to help the students understand that at the very least a eukaryotic organism must be the choice. Once all the students understand a eukaryotic organism is realistically the only choice, continue the discussion by asking the students their thoughts (based on prerequisite material, previous lectures in the course, and mainly from the assigned reading) on which types of eukaryotic organisms or cells seem to make the best candidates for answering this challenge. Have the students form groups and ask them to report the group s choice. Their report must be supported with rationale and a defense. Help the students realize that product quality and safety can be attained from mammalian organisms or cell cultures since the proteins often undergo post translational modifications to assist in proper folding and

7 glycosylation. This provides the best option for closely mimicking a human protein. Point out to the students therapeutic biologics have been mass produced with good success for the past two decades. This however doesn t mean that the established ways should be followed, because it s not necessarily the optimized ways to achieve high levels of expression and yields at the industrial level. Stress that the organism must be eukaryotic, but help them understand that established processes using cell cultures in a bioreactor aren t necessarily the best thing. Ask the students to consider something other than cell cultures. Lead the discussion on using transgenic plants and even transgenic animals. End the lecture by asking the students to review the assigned literature and come to the next class prepared to discuss the tradeoffs with using transgenic animals and plants compared to bioreactor cell cultures. LECTURE DAY THREE Start off the lecture by reviewing the overall goal of the module. Repeat the challenge question and list the 7 categories developed in the Multiple Perspectives: 1) Molecular biology of different expression systems; 2) Product quality and safety (since it is a recombinant human therapeutic protein need to ensure proper post-translational modifications); 3) Levels of expression and yields (scale up); 4) Manufacturability (production days/year and reactor capacity required); 5) Capital expenditures and production costs; 6) Downstream processing (harvesting of crude protein, product recovery, purification); and 7) Selection of ideal organism. Discuss the readings again with the students and focus on the manufacturability of the therapeutic protein product using different organisms in terms of production days per year and the range of reactor capacity required. Keep the focus on batch sizes, production days per year, batch turnaround and overall throughput. Discuss bioprocesses and biomanufacturing in terms of capital equipment and the overall different production costs for typical commercial scale biologics production. Support this discussion by comparing the economics of different processes and point out the tradeoffs in product quality, expression levels, yields, manufacturability and downstream processing. At this point, the students may already be convinced that transgenic animals are a competitive choice for an organism to answer this challenge. Help them understand what to analyze and how to approach a bioprocess design that will provide the best choice for this particular challenge. Use the information within the assigned readings to make these points. Discuss how a bioprocess engineer s upstream manufacturing design affects downstream purification design and processing. By this point the students will all most likely have transgenic animals as their choice for the organism as the solution to this challenge. Help them understand the impact of harvesting the product, from the milk of transgenic animals, on the downstream processes. Discuss the advantages of this method compared to conventional eukaryotic cell cultures in a

8 bioreactor. Conduct the Test Your Mettle exercise as outlined in the Test Your Mettle section above. Assign the Go Public.