Lesson 3: Explain — Biomanufacturing: An Inquiry Lesson in Growing Cells

Introduction

Biomanufacturing is the process of growing living cells in order to harvest something they produce as a part of their life cycle. These products cannot be produced by chemical means; they can only be produced by the internal machinery of a living cell. Traditionally, wild cultures of organisms were isolated and grown for the production of food or drink – such as: cheeses, sauerkraut, sourdough bread, leavened breads, vinegar, and alcohol. Cells being grown are called a culture.

Today’s biomanufacturing frequently involves organisms that have been engineered. A gene or genes have been inserted into the cell that code for the protein or antibody that is desired as a product. This is an involved process. Scientists must develop a cell line that reproduces well in a culture and keeps that gene as it reproduces and has the proteins that these cells make working the way they are supposed to work. Once a consistent cell line has been developed and tested, they can be grown in quantities large enough to make product to sell to the consumer. This process is ongoing and can take years. Biogen idec in the Research Triangle Park has developed a cell line that makes an antibody that prevents white blood cells from crossing the blood‐brain barrier and attacking the myelin sheath of brain cells. This is what happens when people have the auto‐immune disease, multiple sclerosis. The medicine made by Biogen idec for multiple sclerosis that contains this antibody is called Tysabri.

The cells can be bacteria cells, but sometimes animal or plant cells are needed to make the particular protein you want. Bacterial cells can only translate DNA to RNA to protein and cannot change that protein much once it is constructed on the ribosome. Bacterial cells do not contain the organelles that can change proteins by adding on subgroups to the amino acid chains or by stitching smaller proteins together to make a larger molecule. Proteins have to have the correct three‐dimensional shape to function properly. If their shape isn’t right, they don’t work. Many proteins need further things done to them by the cell after they are translated to give them the right shape. In addition, cells from animals are not easy to grow when they are not in an animal’s body. Cells communicate with each other in the body and respond to signals that tell them to grow or not grow. Tumor cells can ignore these signals, this is why they grow out of control in an animal. Cells that make the product are often blended together with tumor cells (called chimerization) so they will grow outside of a body. The cells used by Biogen idec to make the Tysabri antibody are Chinese hamster ovary cells, or CHO cells.

Part of growing the cells is mixing together a liquid they will grow in, called media. Media preparation must be done in a certain way, with deliberate steps taken at certain times and in such a way that no wild cells will be found growing in the media. If anything other than the desired cells is growing in the media, it is contaminated. Contaminated cultures cannot be used. They can produce poisons that can make people very sick. The cells you are growing will have competition and will not grow as well or may even be killed by the contaminant. Even a small piece of a virus, fungus or wild bacteria can cause death in a patient.

Once good media is made and the right cells are chosen, the cells are added to the media (inoculation) and grown in production quantities. Biogen idec’s production reactor is 15,000 liters. Ajinomoto, another biomanufacturing facility in the Research Triangle, has a 100,000 liter production tank in which they grow a bacterial cell line. But the cells cannot just be dumped from a little vial or flask into this huge tank. They have to be grown in gradually increasing volumes until there are enough cells to go into the tank. This tank is called a fermentation tank or bioreactor. There are many sensors, lines going in and lines coming out of the bioreactor. They monitor things like pH, amounts of dissolved oxygen and cell numbers. Lines can bring in nutrients for the cells, acids or bases to adjust pH, and gases. Everything must stay free of contamination. It must be sterile, with absolutely no viruses or organisms growing – except for the production cells.

Once the cells are added to the media, they initially grow very slowly (Lag Phase). Once they become accustomed to their environment, they begin to reproduce very fast (Log Phase). As the cells become more crowded in the bioreactor, the growth slows down (Deceleration) and levels off (Stationary). Eventually, cell numbers decrease as cells start to die off. The product is often harvested around the deceleration phase by removing the cells and media from the bioreactor. The gradual isolation of the product from this mixture of media, cells, their wastes and debris is called downstream processing.

Downstream processing starts with the removal of cells from the media, called recovery. Two ways cells can be removed by using a centrifuge or by filtration. Next is purification. The media goes through a series of steps that gradually eliminate any impurities and result in a pure product in solution. Proteins and antibodies are sensitive to temperature, pH and high concentrations of ions – so only certain methods or purification can be used. Column chromatography is one method that is very commonly used. The liquid passes through a column with tiny beads that bind to the product. A solvent is then passed through the column and it washes the product out, dissolved in the new solvent. After the product is purified, it is put in the form the customer gets and packaged. These steps are called: formulation, filling and packaging. Talecris in Clayton does their formulation and filling on site. Areas where this is done must be extremely clean, especially if the product is injectable – to be put directly into a person’s bloodstream. Contamination, even at this step, could result in a patient’s death. Areas where filling takes place are strictly regulated, as is the entire process.

This is all very different than regular chemical manufacturing. The process is dependent upon a living thing, not just on the scientist’s ability to create a chemical reaction. The steps take longer and cost a great deal more than other types of manufacturing. The benefit is that you can create targeted treatments that act directly on a very specific molecule, unlike synthetic chemicals which have a very general action and can have unforeseen consequences.

Learning Outcomes

Students will complete a cell growth activity designed to allow them to understand the intricacy of biomanufacturing pharmaceuticals in contrast to chemical manufacturing. Students will conduct an inquiry investigation of factors that affect cell growth and explain their results. They will grow yogurt bacteria in milk media to try to produce lactic acid. They will have the option of adjusting variables in media preparation or during fermentation to try to optimize cell growth and the amount of product produced. Cell growth will be assessed by a serial dilution of media and growth on an agar plate of one drop of this dilution. Product production will be evaluated using a change in pH.

Curriculum Alignment

• Goal 7.05 NCSCOS: Investigate aspects of biotechnology including: Specific genetic information available. Careers. Economic benefits to North Carolina. Ethical issues. Impact for agriculture.
• 6.01 Cell function is similar in all living things.
• 7.01 Compare and Contrast microbes
• 7.03 Calculate the reproductive potential of bacteria
• 4.01 Understand that both naturally occurring and synthetic substances are chemicals
• 4.05 Identify substances based on characteristic chemical and physical properties: chemical reactivity (pH and catalase), solubility (milk proteins)
• 4.06 Describe and measure quantities related to chemical and physical changes within a system. Precipitate, Gas Production
• 4.09 Describe factors that determine the effects a chemical has on an organism: exposure, potency, dose, concentration, susceptibility.
• 4.10 Describe Risks and Benefits of chemicals including: medicines, food preservatives and sanitation

Classroom Time Required

Day	1	2	3	4	5	6	7
Activity	Aspirin Demonstration Instruction and Student Planning	Media Preparation	Labeling, Inoculation, Testing pH	Testing pH, Observing cells under microscope, serial dilution of cells, plate inoculation	Plate Observation	Plate Observation, Catalase Testing, Data Analysis, Report Preparation	Report presentations, Class Discussion
Time Req’d	45 minutes	40 min	30 min	60 min	10 min	60 min	15‐30 min.

Class time used can be reduced by: the teacher preparing the media, eliminating the cell counting and serial dilutions with plates, and having students complete more of their reports at home.

This lesson could be extended by: gram staining bacteria from the colonies, growing a monoculture from a plated colony and comparing results to the initial cell growth experiment, attempting to filter the cells and coagulated proteins from the media, an isolation using a chromatography column.

Materials Needed

For cell growth: Dehydrated skim milk; Wyler’s reduced sodium bouillon cubes; corn syrup; yogurt bacteria cells (available from Cheese Making supply, or grocery store yogurt with live cultures); pH test (phenol red, litmus powder, pH strips); tubes or bottles to grow liquid cultures in; ; 70 percent alcohol or 10 percent bleach; pipettes or droppers; weighing balance; slides; cover slips; microscope;

For plating: premade agar plates OR dry nutrient agar and petri dishes; sterile swabs,inoculating loops, toothpicks or glass rods

For aspirin synthesis demonstration: 600 mL beaker; salicylic acid (2 g per demo); acetic anhydride (5mL per demo); 50 mL Erlenmeyer flask; 85 percent phosphoric acid (5 drops per demo); hot water bath; distilled water; ice bath

Optional: Gram stain kit; autoclave or pressure cooker; incubator; filter paper, cell counting slide (hemacytometer, cell‐ometer, or grid slide; microscopes with 100x objectives; slide clamps; skim milk agar; self‐contained LPG burner.

Technology Resources

A microscope camera would be a fun addition to this activity. Students could use presentation or publishing software to create a presentation product on their experiment and results.

Pre‐Activities

Before this lesson, students should be familiar with microscope use, microbiology safety, cell structure and function and microorganisms.

Activities

1. Aspirin Demonstration
2. PowerPoint‐ Lecture on Biomanufacturing
3. Inquiry on Growing Living Cells
   
   1. Making Media
   2. Monitoring Cell Growth
   3. Appraisal of Cell Products

Extensions:

1. Checks for Contamination
2. Gram Staining
3. Isolation and Growth of a Monoculture
4. Recovery via Filtration
5. Chromatography Columns

Assessment

Students will present laboratory reports to the class. A rubric will be used to evaluate performance on the inquiry assignment.

Modifications

Students could report out as a group, turning in one written report. Then, students could be delegated (or delegate themselves) to tasks that allow them to work to their strengths. The report could be in presentation poster form, where each group member is responsible for a different part of creating or presenting that poster.

Alternative Assessments

Students could present in power point, create a video teaching someone how to do one of their activities or write a paper describing what was done and how it could be improved.

Critical Vocabulary

• Bioreactor
• Cell
• Contamination
• Culture
• Fermentation
• Gene
• Inoculation
• Protein
• Media

Websites

• www.ncbiotechcenter.org

Comments

All components of this lesson should be completed to give students a complete understanding of the complexities involved in biomanufacturing.