Bioprocessing Part 3: Purification

Summary

This video explores the essential biotechnology processes behind the purification of Green Fluorescent Protein (GFP), a widely-used biological marker. The purification process is detailed through fermentation, recovery, and purification steps, focusing on techniques like chromatography and tangential flow filtration (TFF). Viewers will learn how chromatography separates proteins based on size, charge, and hydrophobicity, while TFF concentrates and cleanses the GFP. The video also covers the equipment, sensors, and steps involved in producing a highly purified GFP solution for biopharmaceutical use.

Takeaways

• 🔬 Biotechnology plays a significant role in providing essentials like food, clothing, fuel, pharmaceuticals, and eco-friendly plastics.
• 🧫 The three core biotechnology processes are fermentation, recovery, and purification, which are key to producing various products.
• 🧪 GFP (Green Fluorescent Protein) is commonly used as a biological marker due to its non-interference with normal cell function.
• 📊 Chromatography and filtration are the primary operations used during purification, focusing on separating target proteins like GFP.
• ⚗️ Chromatography relies on physical differences such as size, charge, and hydrophobicity to separate proteins.
• 🌡️ Ion-exchange chromatography separates molecules based on charge, while hydrophobic-interaction chromatography uses differences in water solubility.
• 💧 Tangential Flow Filtration (TFF) is more efficient than conventional filtration, using cross-flow to prevent filter clogging.
• 🔍 The purification process includes multiple steps like chromatography, concentration, diafiltration, and final filtration to purify GFP.
• ⚙️ Sensors like UV, conductivity, and pressure help monitor and control the purification process by providing real-time data.
• 🧼 The entire purification area and equipment must be cleaned, disinfected, and prepared meticulously before the process begins.

Q & A

What are the three core biotechnology processes mentioned in the video?

The three core biotechnology processes are Fermentation, Recovery, and Purification.

What is the purpose of Fermentation in biotechnology?

Fermentation is essentially 'cell farming,' where cells are programmed to produce a desired product, grown, nurtured, and then harvested.

How does the Recovery process work in biotechnology?

In Recovery, the product is separated from the cells where it was housed, preparing it for further purification.

What is Purification, and why is it important?

Purification removes any remaining contaminants from the product, leaving a highly pure and concentrated solution. It's crucial for ensuring the final product meets quality standards.

What protein is being purified in the process described, and why is it important?

The protein being purified is GFP (Green Fluorescent Protein), which is widely used as a biological marker because it is well-tolerated by cells and doesn't interfere with their normal functions.

What are the two main operations used in Purification, as discussed in the video?

The two main operations in Purification are Chromatography and Filtration.

How does size-exclusion chromatography work?

Size-exclusion chromatography uses beads with small holes to trap or slow down smaller molecules as they move through the column, while larger molecules pass through more quickly.

What is the principle behind ion-exchange chromatography?

Ion-exchange chromatography works by using oppositely charged beads to attract and bind charged molecules from the process stream.

What is Tangential Flow Filtration (TFF), and how does it differ from traditional filtration?

TFF is a filtration method where fluid moves tangentially across the filter membrane rather than directly into it. This helps reduce clogging and allows for more efficient filtering and recirculation.

What are the main steps involved in the purification of GFP?

The steps include Anion-Exchange Chromatography, Hydrophobic Interaction Chromatography (HIC), Tangential Flow Filtration (TFF), and final filtration, with each stage progressively purifying and concentrating the GFP.