Bioprocessing Part 2: Separation / Recovery

Summary

The video explains the fermentation and recovery processes used in biotechnology, focusing on the production of Green Fluorescent Protein (GFP). It outlines the steps of intracellular fermentation, where the cells are broken open to release the product, and the subsequent recovery process. This involves separating cells from the broth, washing, lysing, and using centrifugation and filtration to isolate the GFP. The clarified lysate containing GFP is then sent for purification. The video highlights the tools and methods used, including centrifuges, homogenizers, and filters, to ensure efficient product recovery.

Takeaways

• 🦠 Many biotechnology companies use fermentation to produce valuable compounds via microorganisms.
• 🔄 Fermentation allows the production of molecules on a large scale using millions of microbial cells.
• 🌱 Two types of production in fermentation: extracellular (product secreted by cells) and intracellular (product kept inside the cells).
• 💧 Extracellular production involves keeping the medium with the product, while intracellular requires cell disruption to release the product.
• ⚙️ The recovery process follows fermentation and involves separating cells from the liquid broth and isolating the molecule of interest.
• 🧬 Tools like centrifuges, homogenizers, and microfiltration are used to extract the product from cells.
• 🔬 Green Fluorescent Protein (GFP) is used as a biological marker to track drug movements in cells.
• 💡 Recovery steps include centrifugation, cell washing, homogenization (cell breaking), and filtration to separate cell debris and collect the product.
• ⚗️ The product (GFP) is purified and concentrated after recovery to ensure stability and remove impurities.
• 📜 The entire process is documented in a Batch Process Record (BPR) for traceability and verification.

Q & A

What is the main purpose of fermentation in biotechnology?

The main purpose of fermentation in biotechnology is to use microorganisms to produce valuable compounds or products on a commercial scale. This allows for the production of large quantities of a molecule of interest by using microorganisms as tiny micro-factories.

What are the two types of production in fermentation, and how do they differ?

The two types of production in fermentation are extracellular and intracellular production. In extracellular production, the product is secreted by the cells and separated from the biomass early in the process. In intracellular production, the product remains inside the cells and is released by disrupting the cells during separation.

What is the role of Recovery in the fermentation process?

Recovery is the step following fermentation, where the cells containing the product of interest are separated from the liquid broth, and the product is extracted from the host cells. It involves separating cells from the broth, breaking them open to release the product, and removing any cell debris.

Why is GFP (Green Fluorescent Protein) used in biotechnology, and what is its role?

GFP is used as a biological marker in biotechnology. When attached to a drug, it provides a visual marker that allows researchers to track where the drug goes in the body. GFP is fluorescent and does not interfere with normal cellular functions, making it ideal for research purposes.

What are the key tools used in the GFP Recovery process?

The key tools used in the GFP Recovery process are a Disc Stack Centrifuge for separating solids from liquids, a Homogenizer to break open E.coli cells, and a 0.22 micron filter to remove any remaining solids from the solution.

What is the purpose of centrifugation in the Recovery process?

The purpose of centrifugation in the Recovery process is to separate solids (the E.coli cells) from the liquid broth. The denser solids are forced to the sides of the centrifuge bowl, while the clarified liquid exits. After the cells are broken open, centrifugation is used again to separate cell debris from the desired product.

What happens during the homogenization step in Recovery?

During homogenization, the cells are forced through a small orifice at high pressure, causing them to rupture and release their internal contents, including the desired product, GFP. The process is repeated to ensure all cells are disrupted.

How is cell debris removed after the cells are lysed in Recovery?

After the cells are lysed, the mixture of cytoplasm and cell contents is centrifuged again to remove the solid cell debris. The liquid, which contains the GFP, is kept, and the remaining solids are discarded.

What is the purpose of filtration in the final step of Recovery?

The purpose of filtration is to remove any remaining small particles or solid materials from the lysate. A 0.22 micron filter is used to ensure the product solution is free of solid contaminants before it moves to the Purification phase.

What happens to the product after the Recovery process is completed?

Once Recovery is complete, the clarified lysate containing the GFP is sent to the Purification step. During Purification, dissolved impurities are removed, and the GFP is concentrated and stabilized for further use.

Outlines

00:00

🧬 Understanding Fermentation and Recovery in Biotechnology

This paragraph explains how biotechnology companies use fermentation to scale up the production of valuable compounds using microorganisms. It distinguishes between extracellular and intracellular production, focusing on the latter where the product remains inside the cell. The paragraph then shifts to the downstream process of Recovery, highlighting the separation of cells from the liquid broth and the isolation of the target molecules. Tools such as centrifuges, cell disrupters, and microfiltration are introduced as key components of Recovery, specifically for producing Green Fluorescent Protein (GFP), a biological marker used in research. The process includes multiple stages like harvesting, separation, and cell disruption, leading up to the purification phase.

05:02

📋 Step-by-Step Guide to GFP Recovery Process

This paragraph details the structured approach required in the GFP Recovery process, emphasizing the importance of meticulous documentation through the Batch Process Record (BPR). Each step must be verified by operators, with specific requirements for cleanliness and equipment setup according to Standard Operating Procedures (SOPs). The GFP Recovery starts with the broth tank's arrival and uses a Disk-Stack Centrifuge to separate solids (E.coli cells) from liquids. The process includes steps such as washing the cells, using a Homogenizer for cell disruption, and ensuring that all cells rupture under high pressure. The centrifuge is then used again to separate the cell debris from the GFP-containing liquid, called Lysate.

10:05

🔬 Final Filtration and Preparation for Purification

The final paragraph covers the last stage of the GFP Recovery process, focusing on the filtration of the Lysate. The centrifuge Lysate is passed through a 0.22 micron filter to remove any remaining solid materials, resulting in Clarified Lysate. This marks the end of the Recovery process. The clarified Lysate is then transferred to a controlled vessel, ready for the next phase of production: Purification. This stage involves removing dissolved impurities and concentrating the GFP, preparing it for its final application in research and diagnostics.

Keywords

💡Fermentation

Fermentation is a biological process where microorganisms such as bacteria or yeast are used to produce valuable compounds like amino acids, enzymes, or drugs on a large scale. In the video, fermentation is highlighted as the initial stage in biotechnology production, where the microorganism E.coli is used to create the desired product, Green Fluorescent Protein (GFP).

💡Intracellular production

Intracellular production refers to the process where the product of interest is retained inside the cell. During recovery, the biomass containing these cells is collected and disrupted to release the product. The video focuses on this type of production for GFP, where the cells are broken open to access the protein.

💡Extracellular production

Extracellular production occurs when the product is secreted by the cells into the surrounding medium. In this case, the cells are discarded, and the medium containing the product is processed. The video contrasts this with intracellular production, explaining that for GFP, the product is not secreted but kept inside the cells.

💡Recovery process

The Recovery process involves separating the product of interest from the host cells and the surrounding medium after fermentation. It includes steps such as centrifugation, cell disruption, and filtration. In the video, the Recovery process is detailed as crucial for extracting GFP from E.coli cells before moving on to Purification.

💡Centrifugation

Centrifugation is a method used to separate components of a mixture based on density by spinning at high speeds. The denser materials, such as cells, move to the sides of the centrifuge bowl, while the liquid is separated out. The video shows centrifugation as a key step in separating E.coli cells from the liquid broth during the GFP Recovery process.

💡Homogenization

Homogenization is the process of breaking down cells to release their contents. In the video, it is used to rupture E.coli cells by forcing them through a small orifice at high pressure. This is a critical step for GFP recovery, as it ensures the protein is released into the solution from the host cells.

💡Green Fluorescent Protein (GFP)

GFP is a protein used as a biological marker to track cellular processes. It fluoresces under specific lighting, allowing researchers to observe the location and behavior of molecules within cells. The video focuses on the recovery process of GFP from E.coli, highlighting its utility in scientific research.

💡Batch Process Record (BPR)

A Batch Process Record is a document used to track each step of a production process, ensuring compliance and accuracy. It requires verification and documentation at each stage. The video mentions the BPR as a guide for operators during the GFP Recovery process, providing a structured framework for operations.

💡Standard Operating Procedures (SOPs)

SOPs are detailed, written instructions to achieve uniformity in performing specific functions. In the video, SOPs are essential for preparing the Recovery area, ensuring equipment is cleaned, sanitized, and set up according to predefined standards before beginning the GFP Recovery process.

💡Clarified stream

The clarified stream is the liquid that exits the centrifuge after most of the solids have been removed. In the context of the video, it is described as containing very few cells after centrifugation. However, this liquid is discarded because the target product, GFP, is still within the E.coli cells, which are collected for further processing.

Highlights

Biotechnology companies utilize fermentation to produce valuable compounds on a commercial scale.

Fermentation scales production from a single cell to millions of micro-factories producing various products.

Extracellular production involves cells secreting products like amino acids, enzymes, or monoclonal antibodies.

Intracellular production keeps the product inside the cells, requiring biomass collection and cell disruption for release.

Recovery process separates cells from the liquid broth and extracts molecules of interest.

Tools like centrifuges, cell disrupters, and microfiltration are crucial for product recovery.

Green Fluorescent Protein (GFP) is used as a biological marker to trace drug movement in research.

The recovery process includes separating cells, washing them, and breaking them open via homogenization.

After homogenization, centrifugation removes cell debris, isolating the desired GFP in the clarified liquid (lysate).

A final filtration step through a 0.22-micron filter removes any remaining solid material.

The clarified lysate is transferred to purification, where dissolved impurities are removed and GFP is concentrated.

Disc Stack Centrifuges separate solids from liquids during recovery.

The process relies on a Batch Process Record (BPR) to guide operators step-by-step with documentation for accuracy.

Homogenizers rupture E. coli cells by forcing them through a tiny orifice at high pressure (900 bar).

Purification stabilizes and refines GFP after recovery, preparing it for further applications.

Transcripts

Many of today's biotechnology companies rely on fermentation to produce their products.

In the typical process, a particular microorganism is used to produce a valuable compound or product.

Fermentation permits us to scale-up that production.

Instead of a single cell producing our "molecule of interest", we use hundreds, thousands and

then millions and millions of these tiny micro-factories to produce many different products on a commercial scale.

In fermentation, there are two ways in which the product of interest is produced by the cells...

Extracellular production is when the cell "secretes" the product of interest.

Examples of this could be certain amino acids, enzymes or monoclonal antibodies....

In the case of extracellular production, the cells -- or biomass - are discarded at the

beginning of separation and the medium -- which contains the product - is kept for further processing

Intracellular production is when the product

of interest produced by the cells is "kept" inside the cell...

and during the early stages of separation, the biomass is collected and then disrupted

so that the product is released.

In our treatment of fermentation, we concentrate on intracellular fermentation.

But fermentation is only the beginning of this journey. Let's look downstream at the next process step in the production flow - Recovery!

The final product of fermentation is called broth. This broth contains the "molecule of interest".

However the molecule is still locked inside a host cell... and millions of these host cells are suspended

in a pool of depleted media and metabolic waste products.

In the next step, Recovery, we perform two important functions;

separating the cells -- the solids in our broth - from the liquid...

And then separating the molecules of interest from their host cells.

Exactly where within the production flow those technologies are

employed varies significantly from product to product.

Recovery processes commonly use a variety of tools - such as centrifuges, cell disrupters, and microfiltration - to isolate the product.

In this program, we are going to look at a typical recovery process used in the manufacture of GFP - (Green Fluorescent Protein).

GFP is broadly used as a biological marker which - if attached to a drug - could provide

researchers with a visual story of where the drug goes. It's a fluorescent dye that's very

well tolerated by most cells and doesn't interfere with normal cellular function.

So how does Recovery work - and how does it fit into the total production flow?

Here's a very basic overview. After Fermentation is complete, the broth

is harvested and sent to Recovery. Here the E.coli host cells are separated from

the liquid broth... Then suspended in a new solution to wash the cells...

Separated again...

Resuspended in a new solution... Homogenized to break open the cells...

The cell debris is removed... Once Recovery is complete, the product is

sent to Purification to be refined and concentrated.

But to really understand Recovery, we'll need to closely examine our process so we can appreciate what each step accomplishes,

and why it's important to the process.

Let's start with the tools we'll use in the Fluorescent Green Protein Recovery process.

Those would include a Disc Stack Centrifuge to separate solids from liquids...

A Homogenizer to break open the E.coli cells...

And a.0.22 micron filter to separate any remaining solids in the product solution.

Our materials include the Broth from our fermentation process...

High Purity Water that has been reverse-osmosis filtered, de-ionized and UV sterilized...

and a buffering solution to help stabilize the pH of our product and keep it in suspension and prevent the product from degrading.

The Recovery process is managed through the use of a Batch Process Record (BPR).

The Batch Record leads the operator through the process, step-by-step...

with each step requiring a sign-off and separate verification by a second operator.

This record also includes spaces for documenting times, activities, operation steps, and instrument readings.

Before the process can begin, the Recovery area must be cleaned and organized.

Any unnecessary equipment or materials should be removed...

And the area must be cleaned and disinfected to decrease the level of microorganisms.

All equipment must be cleaned, sanitized, and set up as required by Standard Operating Procedures (SOPs)...

All required materials and documentation must be gathered and prepared...

And any updates to the Process Control software should be made and verified.

GFP Recovery begins with the arrival of the broth tank.

A sterile hose is run from the broth tank to the Disk-Stack Centrifuge...

and the tank is pressurized to drive the broth into the centrifuge.

After the centrifuge has reached a stable running speed...

the inlet valve is opened and broth enters the bowl.

The centrifugal force of the rotation forces the denser material -- the solids - to the sides of the bowl

(much like the spin cycle on your clothes washer!), while the liquid flows through and out of the centrifuge.

As more broth enters the bowl, it displaces the now clarified (solids removed) liquid

to the top of the bowl where it exits...

while the cells continue to build up on the bowl surface.

The centrifuge has an integrated RPM monitor.

If the unit is not rotating at a stable running speed, the controller will alarm and shut it down.

The liquid leaving the bowl is known as the "clarified stream", because almost all the solids have been removed.

A sensor monitors the clarified stream for "percent solids".

When this value rises it indicates that the bowl is at capacity, and the solids must be removed before processing more broth.

The solids are the E.coli cells, and they contain the product...

When the bowl has reached capacity for solids,

the bowl opens and the solids are discharged into an appropriate container for collection.

Once the solids are discharged, the centrifugation step can resume...

while the clarified liquid is waste. At this point, the cells are in a paste form,

and although most of the liquid has been removed, our cell paste is still about 40% liquid weight.

The remaining liquid contains high levels

of metabolites and salts that could complicate downstream processing, so we're going to lower those levels by "washing" the cells.

The cell paste is suspended in a buffered solution...

and then run through the centrifuge again.

As the clarified liquid leaves the centrifuge this time, it carries many of the contaminants

from the fermentation step with it.

The cells, once again in paste form, are ready for the next step - Cell Disruption, also called Lysing.

The cells are resuspended in a buffered solution... and then pumped at high pressure, 900 bar

- which is about 13,000 pounds per square inch - through the Homogenizer.

Inside the Homogenizer they are forced through a tiny orifice.

Just like a balloon being tightly squeezed, the cells can't take the stress - and they rupture and break apart.

And to ensure that all the E.coli cells are ruptured, the solution is cycled through the homogenizer a second time.

After the second homogenization, the lysed cell solution is pumped back through the centrifuge.

But this time, our goal is different!

Before lysing, our product was held within the E.coli cells.

Now -- with the cells broken apart, the cell contents - including cytoplasm and green fluorescent protein

- are mixed into the buffered solution. The centrifuge again spins out the solids

- which are primarily cell debris - and it's the clarified liquid which contains

our product: GFP! This time we discard the solids and keep the

liquid -- which is now known as Lysate!

Although the centrifuge has removed almost all of the cell debris, some small particles still remain.

We'll remove those with our final Recovery process step: Filtration.

The centrifuge lysate is pumped through a 0.22 micron filter.

This filter is fine enough that it removes virtually all of the remaining solid materials.

At this point, the process stream is referred to as Clarified Lysate.

The Recovery process is finished.

The Clarified Lysate is pumped into a vented, temperature-controlled transfer vessel.

This Lysate tank then moves downstream to the next series of process steps--

Purification--where dissolved impurities are removed from the GFP solution, and GFP is subsequently concentrated and stabilized.