Downstream Processing

Question 1

What is downstream processing?

Answer 1

Downstream processing is the process of extracting the protein out of the bacteria, purifying it and getting it into the market

Question 2

What steps are in downstream processing?

Answer 2

• Cell banks
• Cell Lysis methods
• Protein purification and Chromatography
• Concentration
• Product Formulation
• Product Filling, sealing and packagin

Question 3

Draw a diagram to show where your protein product would end up in the cell

Answer 3

week 6 session 5b slide 4

Question 4

How would you separate your proteins?

Answer 4

This depends on where the recombinant proteins is located

Intracellular cells (inclusion bodies, cytoplasm)
- you want to keep the cells
- centrifugation/ filtration
- cell disruption

Extracellular (in the media)
- you want to get rid of the cells since the protein is in the media
- centrifugation/ filtration
- Bacteria are easily sedimented above 6000xg

Question 5

What is cell disruption?

Answer 5

cell disruption is the process of breaking open (otherwise known as lysing) cells in order to obtain the intracellular fluid (commonly referred to as lysate).

Question 6

What are the ways of lysing E.coli?

Answer 6

Chemical:
- Freeze thaw (inefficient)
- Detergent (often denature the protein)
- Lysozyme (expensive)

Mechanical:
- Ultra- Sonication
- French press
- Bead beater
- Homogenisation

Question 7

What is French Press lysing?

Answer 7

This is a technique commonly used for lysing bacterial cells, and other microorganisms for the isolation of proteins and other cellular components.

The process involves pressing the plunger down through the cylinder where the cell suspension is forced through the orifice and into the jet where the cells break open open into the impact plate.

Question 8

What is ultracentrifugation?

Answer 8

It is a method of separating the cell debris that was in the media after the lysis of the cell if the protein was in the cell or the cell in the media.

Question 9

How is ultracentrifugation used to lyse eukaryotic cells?

Answer 9

1. Filter homogenate to remove clumps of unbroken cells, connective tissue etc.
2. The filtered homogenate is spun at 600 g x (acceleration), 10 mins
3. After 10 mins is done, you should get a pellet of an organelle. (biggest to smallest.
4. Pour out the homogenate that was left over into a clean tube and repeat steps 2 and 3 but at a higher speed and different time period.

Question 10

Draw the steps of Ultracetrifugation of lysed eukaryotic cells.

Answer 10

Week 6 session 5b slide 8

Question 11

How are bacteria centrifugated?

Answer 11

Bacteria have no organelles, therefore centrifugation is simpler.

Whole bacteria
–> Lysed at 3000-10,000 g, 10mins
Bacterial membranes/ debris sedimented
–> +150,000g, 1h
The supernatant = bacterial cytosol often containing the protein of interest

Question 12

What is the difference between centrifugation and ultracentrifugation?

Answer 12

The key difference between centrifugation and ultracentrifugation is that centrifugation uses a low speed for the separation process, whereas ultracentrifugation uses a high speed for the separation process.

Question 13

What is ultrasonication cell lysis?

Answer 13

Sonication is the process of using sound energy greater than 20 kHz (ultrasonic) to cause air bubbles in a liquid to implode in a process called cavitation. In sonication cell lysis the energy released from cavitation impacts the cell membrane and the membrane is irreparably damaged

Sonication is performed either in a water bath or by a sonication probe. When using a probe it is important to ensure that the size of the probe is matched the sample volume. Generally, the probe method is preferred for cell lysis because it is easier to keep the sample on the ice during the process. This is important because the heat generated by the vibration is enough to denature proteins if left uncontrolled. A

Question 14

What are the types of filtration of lysates?

Answer 14

Micro-filtration
- Microfiltration is the process of physically removing suspended solids from water, through a membrane. Microfiltration is often used in conjunction with other separation processes such as ultrafiltration and reverse osmosis.
- The filters used in microfiltration have a pore size of approximately 0.1 micron (small). Bacteria and suspended solids are the only element that can be removed through microfiltration.

Ultra-Filtration
- Ultrafiltration blocks everything microfiltration can with the addition of viruses, requiring a slightly higher pressure to achieve this.
- Although it requires higher pressure than MF, ultrafiltration can be powered by the pressure you get from the tap, making it popular in the commercial sector for drinking water.
- It works the same way as MF by which a contaminated liquid passes through a membrane that is too large to fit through the membranes pore sizes, yielding a purified liquid stream.
- Ultrafiltration filters have a pore size of approximately 0.01 micron (smaller).

Question 15

Draw a workflow of the lysis and centrifugation

Answer 15

Week 6 session 5b slide 11

Question 16

What are inclusion bodies?

Answer 16

• Many human proteins are expressed in E. coli but most form inclusion bodies
• Inclusion bodies are insoluble aggregates of partially folded heterologous protein
• IBs formed in the cytoplasm
• Almost all of inclusion body protein is the heterologous protein of interest (highly pure)
• Because of their dense nature, they are easily separated by centrifugation.
• But protein must then be renatured and re-folded

Question 17

Advantages of Inclusion bodies

Answer 17

• Separation easy
• Very high purity
• Not degraded by proteases
• Protein is not functional – so not toxic

Question 18

How do you isolate inclusion bodies and renature proteins?

Answer 18

1. Lyse cells using a method of choice
2. Perform a low-speed spin (centrifugation) after cell lysis
3. Wash the pellet and resolubilized the protein with 8M Urea
4. Remove Urea by filtration or dialysis

Question 19

How do you isolate inclusion bodies and renature proteins?

Answer 19

1. Lyse cells using a method of choice
2. Perform a low-speed spin (centrifugation) after cell lysis
3. Wash the pellet and resolubilized the protein with 8M Urea
4. Remove Urea by filtration or dialysis

Question 20

How would you purify proteins?

Answer 20

Ultracentrifugation and ultrafiltration may have already removed most contaminating proteins but column chromatography makes sure to separate pure proteins

Question 21

What is Column chromatography?

Answer 21

It is a precursory technique used in the purification of compounds based on their hydrophobicity or polarity.

Separates molecules depending on how they interact with a solid stationary phase (the chromatographic beads) and a mobile phase (usually a buffer).

The beads are packed into the glass column.

Question 22

Draw and label the system for column chromatography

Answer 22

Week 6 session 5b slide 20

Question 23

What are the different types of chromatography and their uses?

Answer 23

• Ion-exchange chromatography - the difference in protein surface charge at a given pH
• Gel-filtration chromatography - Difference in mass/shape of different proteins
• Affinity chromatography - Based upon bio-specific interaction between a protein and an appropriate ligand
• Hydrophobic interaction chromatography - Difference in surface hydrophobicity of proteins
• Chromatofocusing - Separates proteins on the basis of their isoelectric points
• Hydroxyapatite chromatography - Complex interaction between proteins and the calcium phosphate-base media, not fully understood.

Question 24

What is Size Exclusion Gel Filtration Purification?

Answer 24

• Size-exclusion chromatography also called gel filtration, separates proteins according to size.

-The degree of cross-linking controls the average pore size of the gel prepared. Most gels synthesized from any one polymer type are thus available in a variety of pore sizes.

• The higher the degree of cross-linking introduced, the smaller the average pore size and the more rigid the resultant gel bead. Highly cross-linked gel matrices have pore sizes that exclude all proteins from entering the gel matrix
• Larger proteins migrate faster than smaller ones and take a more direct route through the column.
• The smaller proteins enter the pores and are slowed by their more labyrinthine path through the column.
• Size-Exclusion is used more in academia than in industry, and more at the polishing step because it’s difficult to get it right at a larger scale.

Question 25

What is Affinity Purification?

Answer 25

• Affinity chromatography separates proteins by their binding specificities.
• Bind specifically to a ligand cross-linked to the beads.
• After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the column) by a solution containing free ligand.
• Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification can be quite high depending on the specificity of the interaction and, consequently, it is generally the first step, if not the only step, in a purification strategy.

Question 26

What is IMAC?

Answer 26

Immobilized metal affinity chromatography (IMAC) is a protein separation method based on the interaction between proteins in the solution and transition metal ions fixed to a solid support.

Question 27

Why are IMAC good for purification?

Answer 27

Histidines and cysteines, when presents are the most important metal-coordinating residues on the protein surface.

Histidine tags bind strongly to nickel beads. Bound his-tagged proteins are eluted with Imadazole

Question 28

What is Lyophilisation?

Answer 28

Lyophilisation or Freeze-drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing ice to change directly from solid to vapour without passing through the liquid phase.

Many freeze-dried proteins can be stored at room temp for long periods of time.

Question 29

What are the pros and cons of using Lyophilization?

Answer 29

Pros:
- Processing a liquid with ease (and thereby simplifying aseptic handling)
- Enhancing the stability of a dry powder as well as the product stability in a dry state
- Removing water without having to heat the product excessively
- Dissolution of the reconstituted product (rapidly and easily)

Cons:
- Handling and processing time increases
- Sterile diluent needed upon reconstitution
- Equipment becomes costly and complex

Question 30

What is Final product filling or Fill & Finish?

Answer 30

It involves:
- Bulking out the product with excipients
- Sterilisation by filtration
- Freeze drying
- Sealing

1. The final bulk product is filter sterilized by passing through a 0.22 um filter.
2. The sterile product is aseptically filled into (pre-sterile) final product containers under grade A laminar flow conditions.
3. Much of the filling operation uses highly automated filling equipment.
4. After filling, the product container is either sealed (by an automated aseptic sealing system), or freeze-dried first, followed by sealing.

Question 31

Draw a flow diagram of the fill & finish process

Answer 31

Week 6 session 5b slide 31

Question 32

What is added to the labelling and packaging of a product?

Answer 32

• name and strength/potency of the product;
• specific batch number of the product;
• date of manufacture and expiry date;
• storage conditions required.