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.

Outlines

00:00

🔬 Introduction to Biotechnology Processes

This paragraph introduces the key role of biotechnology in producing essential products such as food, clothing, fuels, and pharmaceuticals. It explains the three core biotechnology processes—Fermentation, Recovery, and Purification—highlighting their importance in manufacturing products like Green Fluorescent Protein (GFP). The paragraph provides an overview of how these processes work, particularly focusing on the purification process, which involves Chromatography and Filtration.

05:00

🧪 The Role of Hydrophobic Interaction Chromatography (HIC)

This paragraph delves into the details of Hydrophobic Interaction Chromatography (HIC), a method used to purify proteins by exploiting their hydrophobic and hydrophilic properties. It describes how salt is used to remove water shields around hydrophobic patches in proteins, allowing them to interact with resin beads in the chromatography column. The paragraph also highlights the use of HIC and ion-exchange chromatography in the GFP purification process, explaining the components of the chromatography equipment and the sensors used to monitor the process.

10:06

⚗️ Diafiltration and Concentration Techniques

This paragraph explains the processes of Diafiltration and Concentration within the purification of Green Fluorescent Protein. Diafiltration involves exchanging the buffer solution by adding new buffer while removing the old one, which helps in removing unwanted components like salts. Concentration, on the other hand, focuses on removing water and buffer components to achieve a more concentrated solution of GFP. The paragraph also discusses the varying complexity of purification processes depending on the product, ranging from simple to multi-step procedures.

15:07

🧬 Final Steps in GFP Purification

This paragraph outlines the final steps of the GFP purification process, starting with the Anion-Exchange Chromatography, where GFP is separated based on its negative charge. It then describes the Elution process, where GFP is released from the resin beads by introducing a sodium chloride buffer. The paragraph also covers Hydrophobic-Interaction Chromatography (HIC) and Tangential Flow Filtration (TFF) to concentrate and diafilter the GFP solution, eventually leading to the final filtration step where the purified GFP is collected for further processing.

Keywords

💡Biotechnology

Biotechnology refers to the use of living systems and organisms to develop or produce products essential for daily life, such as food, fuel, and medicines. In the video, biotechnology is the foundational theme, specifically in the production of Green Fluorescent Protein (GFP) and other bio-based products using advanced processes like fermentation and purification.

💡Fermentation

Fermentation is a biotechnology process where cells are grown and nurtured to produce a desired product. It’s described in the video as 'cell farming,' where cells are programmed to create specific outputs. The video discusses how fermentation helps in producing materials like GFP before they move on to purification.

💡Green Fluorescent Protein (GFP)

GFP is a protein that emits green fluorescence when exposed to light in the blue to ultraviolet range, commonly used as a biological marker. In the video, GFP serves as the focus of the purification process, showing how it's produced and isolated using chromatography and filtration techniques.

💡Purification

Purification is the process of isolating a single product, like GFP, from a mixture of other cellular components. In the video, purification follows fermentation and recovery, where several methods like chromatography and filtration are used to purify GFP from other proteins and debris in the solution.

💡Chromatography

Chromatography is a purification method that separates molecules based on their physical properties like size, charge, or hydrophobicity. The video explains different types of chromatography used in GFP purification, such as ion-exchange and hydrophobic-interaction chromatography, to achieve high purity levels.

💡Ion-Exchange Chromatography

Ion-exchange chromatography is a technique where charged molecules are separated based on their affinity to oppositely charged beads in a column. In the video, GFP, being negatively charged at a pH of 8.0, binds to positively charged resin, allowing for its separation from other components.

💡Hydrophobic-Interaction Chromatography (HIC)

HIC is a method that separates proteins based on their hydrophobic (water-hating) regions. In the GFP process, high salt concentrations expose hydrophobic patches on the protein, allowing it to bind to resin beads with hydrophobic surfaces, which is later eluted by reducing the salt concentration.

💡Tangential Flow Filtration (TFF)

TFF is a filtration process where the solution flows across a filter membrane instead of directly through it. This method prevents clogging and allows for efficient separation. The video highlights TFF as being used for concentrating the GFP and for diafiltration, a step that exchanges the buffer solution to remove salt.

💡Elution

Elution refers to the process of releasing a bound substance from a chromatography column by washing it with a solution. In the context of GFP purification, elution occurs after binding the GFP to the resin beads, and a buffer with NaCl is used to release the protein into a collected eluate.

💡Centrifugation

Centrifugation is the process of spinning a solution at high speeds to separate solids from liquids based on density. The video explains how centrifugation is used early in the GFP recovery process to remove cell debris and clarify the lysate, leaving a liquid rich in biological components for further purification.

Highlights

Biotechnology plays a key role in the production of food, clothing, fuels, pharmaceuticals, and earth-friendly plastics.

Three core biotechnology processes used in manufacturing are Fermentation, Recovery, and Purification.

Fermentation involves programming cells to produce a product and harvesting them once they grow and reproduce.

In Recovery, the product is separated from the cells where it was produced.

Purification removes contaminants, leaving behind a pure, concentrated solution.

The purification process for Green Fluorescent Protein (GFP) includes two main operations: Chromatography and Filtration.

Chromatography uses physical properties like size and charge to separate target proteins from other molecules.

In size-exclusion chromatography, smaller molecules are trapped by resin beads, while larger ones move faster.

Ion-exchange chromatography separates molecules by charge; opposites attract to charged beads.

Hydrophobic-Interaction Chromatography (HIC) relies on salt to expose hydrophobic protein regions and facilitate interaction with resin.

Tangential Flow Filtration (TFF) is used for concentrating and diafiltering GFP, allowing selective retention of the target protein.

In TFF, the stream moves tangentially across the membrane, reducing clogging and increasing efficiency.

Concentration removes water and buffer components, leaving a more concentrated GFP solution.

Diafiltration involves exchanging the old buffer solution with a new one, removing salts and other unwanted components.

The final step in purification includes passing the GFP solution through a 0.22 micron filter before collecting it for downstream processing.

Transcripts

Today, we rely heavily on biotechnology for many of the necessities of life:

For the food we eat, the clothes we wear, fuels for transportation, earth-friendly plastics,

pharmaceuticals to treat our illnesses, and dietary supplements to help us stay healthy.

Many of these products are produced using three core biotechnology processes: Fermentation,

Recovery and Purification. Fermentation is basically cell farming.

We program cells to produce a product, we nurture them as they grow and reproduce,

and then we harvest them! In recovery we separate our product from the

cells where they were housed... And then in purification we go a step further

by removing everything else that's contaminating our product...

leaving us with a very pure, concentrated solution.

In this program, we're going to look at a typical purification process used in the manufacture

of GFP - Green Fluorescent Protein. GFP is broadly used as a biological marker

because it's very well tolerated by most cells and doesn't interfere with normal cellular

function. We'll examine the technologies, equipment

and materials used... how to prepare for the process...

and how the GFP purification process is managed, step-by-step.

There are two main operations used within purification: Chromatography and Filtration.

Before we can appreciate exactly what these process steps are accomplishing, we need to

take a closer look at what our Green Fluorescent Protein has been through so far.

Back in Recovery, our harvested cells from Fermentation were homogenized.

Our target protein was inside each cell, so to get at it, the cells had to be ruptured.

Homogenization freed a flood of cellular components: including membrane debris, cytoplasm, DNA,

and proteins - including our target protein, GFP -- and they were all mixed into the buffering

solution used to suspend the cells. Most of the solids were then removed through

centrifugation (or "by centrifuge"), but the liquid that remained -- the clarified

lysate, was still rich in biological products, including

dissolved chemicals, proteins and other impurities. Amazingly, through Purification, we can target

a single protein within this biological soup. In our GFP purification process, we'll be

using multiple types of Column Chromatography. The "column" is a cylinder filled with glass,

ceramic or polymeric beads which are engineered to interact with or bind with molecules based

on one or more physical properties. Chromatography relies on differences. Each

molecule has a unique set of physical characteristics; such as size, charge, or extent of interaction

with water. Chromatography uses these differences to separate the target protein from other

proteins and chemicals. Sometimes size is used to differentiate. Some

beads have small holes in them and can temporarily trap or at least slow down smaller molecules

as they travel through the column of resin beads, while molecules too large to enter

the pores move around the beads and exit the column first. This type of chromatography

is called size-exclusion. In the case of charge, opposites attract,

so a negatively charged chromatography bead will attract -- and bind to -- positively

charged components in the process stream. Likewise, a positively charged bead will bind

negatively charged components in the process stream. This charge-based chromatography is

called ion-exchange chromatography. And then there's water. Molecules that readily

interact with and dissolve in water are called hydrophilic (water-loving), while those that

don't are called hydrophobic (water-hating). Proteins contain regions that are hydrophobic

and regions that are hydrophilic. Because water tends to form a shield around the hydrophobic

patches within the proteins, they are not exposed to interact with the resin beads.

By adding salt to the protein solution, we remove the water shield, exposing these hydrophobic

patches on the protein and resin so they can interact. This is how HIC or Hydrophobic-Interaction

Chromatography works. In our process, which uses ion-exchange and

hydrophobic-interaction chromatography, the chromatography equipment is housed on a skid

to make it compact and mobile. The main part of the apparatus is a glass

column filled with resin beads, but we also have...

...pumps to move the clarified lysate through the process...

...a supply hose and port to feed the column... ...a pre-filter to remove any remaining particulates

-- usually solid cell debris that has not previously been removed - before the clarified

lysate enters the column... ...an exit port for the processed solution...

...and auto-switching valves for directing processed solution to either waste or collection.

To help monitor the chromatography equipment - and the solutions flowing through the unit

-- during the process, a number of sensors are located along the product flow path.

There's an electrical-conductivity sensor at the column inlet...

...a pressure sensor just before the pre-filter to help identify a filter clog...

...a flow meter to measure the rate of solution movement through the column...

...and an air sensor to ensure that no air has entered the flow path.

As a solution leaves the column, it passes... ...a UV sensor that reads optical density...

...a second conductivity sensor... ...and a pH sensor that measures how acidic

or basic the solution is. The conductivity sensors let us know when

a new buffered solution has filled the column. When the conductivity reading on the exit

of the column matches the reading from the sensor at the inlet of the column, then we

know the new solution has completely displaced the old one.

The Ultraviolet (UV) sensor monitors the concentration of protein in the product by observing the

optical density of the passing solution. This sensor works hand-in-hand with the valves

on the exit of the column. Through the controller program, we can set

a protein concentration threshold. When the optical density of the solution leaving

the column is below the threshold, the valve directs the flow to waste.

When the optical density of the solution leaving the column is at or above the threshold, this

means that solution contains our purified product...

and the solution is directed to a collection vessel.

TFF - Tangential Flow Filtration - is at its heart, a simple process step.

We're going to pump a fluid through and across a special type of filter known as an ultrafiltration

membrane. The size of the pores in the filter material

determines what passes through and what's held back.

As with almost any filtering process, we can choose what we want to keep.

The solution that passes through the membrane is referred to as the permeate.

Because the pores of the ultrafiltration membrane are small enough to keep the product from

passing through, the permeate contains no product and is sent to waste.

The portion of the feed stream that does not permeate the membrane is called the retentate.

It contains the retained product and is the stream we are most interested in.

What makes TFF different is a core technology that enables it to be faster, more efficient,

more flexible and even self-cleaning! In conventional -- or terminal - filtration,

a fluid is pumped directly into a filter. The particles within the stream that can't

fit through the pores of the filter build up at the filter surface, eventually clogging

it. In Tangential Flow Filtration, the stream

moves across the filter -- that is, tangential to the filter - instead of directly at it.

The cross-flow current actually picks material back out of the filter media or membrane and

into the stream. This retained material -- called retentate

-- is recirculated to the supply tank and will continue to loop through the filter for

as long as the process runs. We'll be using TFF for two different tasks

within the purification process: Concentration and Diafiltration.

We are processing purified GFP from a chromatography step. The fluid from the chromatography step

is purified Green fluorescent protein dissolved in a buffering solution

In Diafiltration, we add new buffer to the GFP retentate, while displacing the old buffer;

effectively exchanging buffer solutions! NOTE: the GFP is retained by the membrane.

If we don't add a new buffer, then we're concentrating our solution.

Concentration is simply the removal of water and buffer components from the feed solution

that results in a more concentrated solution of Green Fluorescent Protein.

From operation to operation or product to product, the purification process can look

quite different. It could be as simple as a single Chromatography

step, one cycle of TFF to concentrate the product, and final filtration...

Or it could involve several different types of Chromatography, Diafiltration between Chromatography

steps, and a final conventional filtration. Our Green Fluorescent Protein process is pretty

typical of the process for biopharmaceutical products:

The clarified lysate will be pre-filtered... Go through Anion Exchange Chromatography...

and the GFP collected and pooled. Next, Ammonium sulfate will be added to make

the solution high-salt... Followed by Hydrophobic Interaction Chromatography-

HIC. The GFP is then collected and pooled again...

The solution then goes through a TFF ultrafiltration step -- which includes concentration of the

GFP followed by Diafiltration to exchange buffer solutions and remove salt.

And the process finishes up with Final Filtration into bulk bottles or bags

It's time to gather everything we'll need for the Purification process and ensure that

the area and equipment are ready to go. The Chromatography skid and TFF system are

checked for proper operation... process hoses are attached and examined for

leaks... We verify that we are using the correct column

resin and that the resin is properly packed. the column is filled with a storage buffer...

and the product path is checked for trapped air and purged if necessary.

Our raw materials include the clarified lysate from the Recovery process,

various buffer solutions that are tailored to specific process steps,

and Ammonium Sulfate that we add to one of the buffers to make it high-salt.

The Purification process is managed through the use of a Batch Record. There is a separate

Batch Record for each processing operation. These documents lead the operator through

the process, step-by-step... with each step requiring a sign-off and separate

verification by a second operator. The Batch Record also includes spaces for

documenting times, activities, operation steps and instrument readings.

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

Any unnecessary equipment or materials are removed...

All equipment must be cleaned, sanitized, and set up as required by Standard Operating

Procedures... All required materials and documentation must

be gathered and prepared...before the process may begin.

The Purification process begins as the transfer tank of clarified lysate from the Recovery

process is connected to the inlet pump on the Chromatography skid.

The first Chromatography step in our Green Fluorescent Protein purification process is

Anion-Exchange. At this point in the process, the pH of the

clarified lysate is about 8.0, which means that the protein is negatively charged. Because

it is negatively charged, GFP will bind to the positively charged anion exchange resin.

The pump draws the lysate from the vessel... past the first conductivity sensor and pressure

sensor... and through the 0.45 micron pre-filter. The

pre-filter removes any residual cell debris or other particulates that may have contaminated

the solution. If the pre-filter begins to clog, the pressure

sensor at the inlet side of the filter will register a rising pressure...

and the controller will signal the need for a filter change.

After pre-filtering and before the column, the lysate passes through...

a flow meter... and an air sensor.

Then, as the lysate passes over the resin beads, the negatively charged protein binds

to the positively-charged beads. The solution leaving the column passes a UV

optical density sensor, a conductivity sensor and a pH sensor.

The optical density sensor's low readings confirm that the GFP is not in the solution,

so the outlet valve sends the solution to waste.

When all the lysate has entered the column -- or when the capacity of the beads to bind

the protein has been reached, it's time for Elution.

Elution is the release of, in this case, Green Fluorescent Protein from the beads by using

a new solution -- in this case a buffer that includes NaCl (sodium chloride) - solution.

As the new buffer is pumped through the beads, at some point the GFP no longer binds to the

beads and is released into the buffer. The resulting product stream is usually referred

to as the eluate (el-you-ate). The UV optical density sensor, which measures

protein concentration, indicates when product begins eluting from the column.

At this point, outlet valves are switched to allow flow of the eluate -- the product

stream -- to a collection vessel. When the UV sensor indicates that all of the

GFP has come off of the chromatography resin, the outlet valves are switched to waste.

When all of the eluate has been collected and pooled, the Anion-Exchange Chromatography

step is finished and it's time for HIC-Hydrophobic-Interaction Chromatography.

Hydrophobic-Interaction Chromatography is based on the principle that hydrophobic chemicals

on the resin surface will bind to hydrophobic patches on the GFP protein.

In order for this to occur, the resin and protein eluate have to be in a high salt environment

to remove the water shielding.  The salt we use is ammonium sulfate.

To remove the attached GFP protein from the HIC column we simply lower the salt concentration

during elution causing the water shielding to reform and the GFP protein detaches from

the resin into the elution stream. The protein-rich eluate is collected and pooled.

The product is now ready for the last major step, TFF.

At this point in the process, Tangential-Flow Filtration will be used to concentrate and

diafilter the GFP product stream. The eluate is rich in Green Fluorescent Protein,

but it's still too dilute... and too high in salt.

As the solution moves through the TFF apparatus, it leaves the supply tank...is pulled through

a pump... past a pressure sensor...

and then across the filter membrane. Everything that passes through the membrane,

including the buffer solution, is known as permeate -- and -- for this process - is sent

to waste. The GFP protein is larger than the pores of

the membrane and therefore is retained. The retained material -- called retentate

-- is recirculated to the supply tank. Recirculation of the feed continues until the desired concentration

of GFP is achieved. Following concentration, and while the protein

solution is recirculating, a new solution -- a storage buffer - is added to the feed.

In effect, the protein is being washed by the flow of a new buffer solution in, and

the old buffer solution out. As this diafiltration step proceeds, the buffer

solution that is being added to the feed replaces the buffer solution that the GFP was originally

in, effectively removing any remaining salt as well.

When this process is complete, the GFP solution is routed through a 0.22

micron final filter... and then collected in appropriate containers

-- usually bottles or bags. The Purification process is complete. The

Green Fluorescent Protein concentrate can now move downstream to final Fill/Finish to

be freeze-dried and packaged.