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BIOL3002 Biotechnology 2: Downstream Processing > LEC 1 SHORT QS > Flashcards

LEC 1 SHORT QS Flashcards

(95 cards)

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1
Q

Q1: What does upstream processing involve?


A

It includes all processes related to growing cells to produce a crude product, from the working cell bank to large-scale bioreactor culture.

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2
Q

Q2: What marks the beginning of downstream processing (DSP)?


A

The harvesting of the crude product.

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3
Q

Q3: Why must upstream conditions consider downstream needs?

A

A: Media components like antifoams may interfere with downstream purification (e.g. ultrafiltration or ion exchange), and organisms should ideally secrete product with low extracellular protease levels.

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4
Q

Q4: How do recovery costs vary by product type?

A

Ethanol: ~15%
Antibiotics: 20–30%
Enzymes and biopharmaceuticals: up to 70%

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5
Q

Q5: What does downstream processing aim to achieve?

A

Separation and purification of a metabolite or biopharmaceutical to a usable purity level, cost-effectively.

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6
Q

Q6: Why is product concentration important in DSP?

A

A: Products are often in dilute solutions — the more dilute the product, the more valuable it usually is and the harder it is to recover.

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7
Q

Q7: How are biotech products categorized based on DSP needs?

A

High volume, low value (e.g. bulk enzymes, amino acids)
Low volume, high value (e.g. biopharmaceuticals, diagnostics)

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8
Q

Q8: How does end-use of a product influence DSP?

A

A: The required purity level varies by end use (e.g. injectable insulin requires ~99.999% purity).

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9
Q

Q9: What factors influence DSP strategy?

A

Product size, stability, chemical nature
Purity and yield targets
Extracellular vs. intracellular location
Biohazards, impurities, market demand

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10
Q

Q10: What is the first major aim in DSP after harvest?

A

A: To reduce the large liquid volumes quickly while achieving some purification and concentrating the product.

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11
Q

Q10: Identify and briefly explain the four major steps in the downstream processing of a biopharmaceutical.


A

Solid-liquid separation (removal of cells)
Dewatering (concentration by precipitation or ultrafiltration)
Purification (chromatography)
Formulation (addition of stabilizers, etc.).

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12
Q

Q1: What is the goal of solid-liquid separation in DSP?

A

To separate cell biomass from the culture broth and retain either the supernatant (for extracellular products) or the cells (for intracellular products or SCP).

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13
Q

Why is temperature control important during this step?

A

To maintain 4°C, which retards biological activity like protease action and bacterial metabolism that could degrade the product.

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14
Q

Q3: Where is the culture usually transferred post-fermentation?

A

A: To a chilled harvest vessel located in a Grade D clean room under GMP.

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15
Q

Q4: What extra step is needed if the product is intracellular?

A

A: Cell disruption to release the product, which adds cost and complexity.

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16
Q

Q5: What factors influence the choice of separation technique?

A

A: Cell size and density, glycocalyx presence, and whether cells form aggregates or pellets.

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17
Q

Q6: What are the two main methods of solid-liquid separation?

A

A: Filtration and centrifugation.

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18
Q

Q7: How does dead-end filtration work?

A

A: Fluid passes perpendicularly through a filter; particles are retained while liquid flows through.

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19
Q

Q8: What factors affect dead-end filtration performance?

A

A: Filter surface area, pore size, pressure, and build-up of solids (filter cake).

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20
Q

Q9: What are common filter media materials?

A

A: Cellulose, glass wool, ceramics, and synthetic membranes.

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21
Q

Q10: What large-scale filters are used for thick broths?

A

A: Rotary drum filters (continuous, ideal for fungi/yeast) and filter presses (batch mode).

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22
Q

Q11: Describe the rotary drum?

A

The rotary drum (0.5 – 3.0 m in diameter) is partially submerged in the culture fluid and as it revolves in the trough it ‘sucks up’ liquid (0.1-2 rpm), leaving the cells as a cake on the surface of a porous fabric.

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23
Q

Q11: Describe the filter press?

A

The filter press comprises a variable number of cloth filters through which a broth may be forced under pressure. The system is cheap, but operates in batch mode (and dismantling causes the filters to wear.)

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24
Q

Q13: What is cross-flow microfiltration and its benefit?

A

A: A method where liquid flows parallel to the membrane surface, reducing clogging; it offers >99.9% cell retention.

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25
Q14: List advantages of filtration over centrifugation.
A: Simpler process, lower power usage, independence from cell/media density.
26
Q13: What is the principle behind centrifugation?
A: Separation based on density differences, using centrifugal force to sediment particles.
27
Q14: What law describes sedimentation in centrifugation?
A: Stoke's Law — sedimentation rate ∝ (particle diameter)².
28
Q15: List some cell types from most to least costly to recover.
Most expensive: Cell debris Less costly: Bacteria, yeast, mammalian, plant cells Least costly: Pellets, flocs, fungal hyphae
29
Q16: What are the main industrial centrifuge designs?
A: Tubular bowl, multi-chamber bowl, disc stack centrifuge, scroll (helical screw) centrifuge.
30
Q17: How does a disc stack centrifuge operate?
A: Culture flows between 20-300 angled discs (35 - 50º and kept 0.4 – 2 mm apart); solids settle on disc surfaces and migrate outward, while clarified liquid exits near the axis.
31
Q18: What are typical forces in disc stack centrifuges?
A: 5,000–15,000 x g
32
Q19: What’s the minimum particle size separated by disc centrifuges?
A: ~5 µm
33
Q20: What is 'polishing' in DSP?
A: A final filtration step to clarify supernatant after centrifugation.
34
Q1: What is broth conditioning?
A: A pre-treatment of culture broth to improve the efficiency of filtration or centrifugation.
35
Q2: What are the main goals of broth conditioning?
A: To modify particle size, reduce particle interactions, and alter viscosity for better separation.
36
Q3: What is a filter aid and how does it work?
A: A slurry of inert, incompressible particles that forms a pre-filter layer, preventing early clogging of the main filter.
37
Q4: Name two common filter aid substances.
A: Diatomite (Dicalite®) and Perlite®.
38
Q5: What is diatomite made from?
A: Fossilized diatom skeletons composed of inert silicon dioxide (SiO₂).
39
Q6: What additional benefit do cationic filter aids provide?
A: They reduce levels of pyrogens, nucleic acids, and acidic proteins, protecting chromatography columns.
40
Q7: Describe the typical procedure for using a filter aid.
a) Apply a thin pre-coat of filter aid to the filter.
b) Mix more filter aid into the broth and begin filtration.
c) Return initial filtrate to the broth before starting full filtration.
d) For vacuum drums, build a thick pre-coat layer before operation.
41
Q8: What is flocculation?
A: A process where particles in broth are induced to clump together into larger aggregates for easier separation.
42
Q9: What substances are used to facilitate flocculation?
A: Polycations — often inorganic salts like ammonium sulfate, sodium sulfate, or alum (aluminium potassium sulfate).
43
Q10: How do polycations promote flocculation?
Neutralize negative cell surface charges Dehydrate the cell surface Expose hydrophobic areas Bridge cells with high-molecular-weight polymers
44
Q11: What natural factors prevent microbial flocculation?
Negative surface charge Hydrophilic cell walls Bound water shell Steric hindrance from irregular cell shapes
45
Q1: Why is cell disruption necessary in some downstream processes?
A: Intracellular proteins (like recombinant insulin) must be released from cells before purification.
46
Q2: What are key concerns during cell disruption?

Maintaining 4°C to reduce enzyme/metabolic activity Minimizing shear damage to sensitive proteins Preventing protease and glycosidase degradation due to broken compartmentation
47
Q3: What factors influence cell wall strength and ease of disruption?
Cell type (Gram-positive/negative, yeast) Culture conditions Growth rate and phase Cell storage method
48
Q4: Which organisms are generally harder to disrupt?
A: Gram-positive bacteria (80% peptidoglycan in their cell walls) and yeasts
49
Q5: How do homogenizers work?
A: Use 40–100 MPa pressure to force cells through a narrow aperture, causing rupture by shear and pressure changes.
50
Q6: What is bead milling (abrasion)?
A: Grinding cells with agitated glass beads (0.2–0.5 mm for bacteria, 0.4–0.7 mm for yeasts) in a wet mill.
51
Q7: What method is commonly used to lyse animal cells?
A: Detergent solubilization — especially with non-ionic detergents like Triton X-100 to reduce denaturation.
52
Q8: Why are non-mechanical methods less suited for large scale?
A: They're less scalable and may complicate downstream purification.
53
Q9: What enzymatic treatments are used for cell lysis?
Lysozyme for Gram-positive bacteria Glucanase, mannanase, protease for yeasts
54
Q10: What are the drawbacks of enzyme use in disruption?
A: Added cost and complexity in downstream purification.
55
Q11: What is ultrasonication and how does it disrupt cells?
A: Uses ultrasonic waves (>18 kHz) to create cavitation — forming and collapsing microbubbles that rupture cells with shockwaves.
56
Q12: Why is ultrasonication limited to lab scale?
A: High cost and heat generation that's difficult to manage at large scale.
57
Q13: After disruption, how is the target product typically separated?
A: Centrifugation to separate cell debris from the biomolecule-containing supernatant.
58
Q1: Why is concentration necessary in downstream processing?

A: The supernatant is typically 85–98% water, and removing this is essential for reducing volume and increasing product yield.
59
Q2: What is the typical product enrichment achieved at this stage?
A: About 2- to 5-fold enrichment of the target biomolecule.
60
Q3: What does membrane filtration achieve?
A: Concentration and limited purification of biomolecules.
61
Q4: How is membrane filtration classified?
A: By pore size and the type of force used (e.g. pressure-driven).
62
Q5: What is ultrafiltration and its typical pore size?
A: A membrane process for concentrating proteins; pore size ranges from 0.001 to 0.02 μm.
63
Q6: What molecular weight cut-offs (MWCO) are common in ultrafiltration membranes?
A: Membranes with 10,000 Da, 30,000 Da cut-offs, etc.
64
Q7: What forces are used to drive filtration?
A: Inert N₂ gas (2–10 bar) or mechanical pumps.
65
Q8: What are the names of the two outputs in membrane filtration?
Retentate: material retained by the membrane Filtrate: liquid that passes through
66
Q9: What materials are used to make membrane filters?
A: Ceramics, steel, PVC, polyvinylidene fluoride, and polycarbonate.
67
Q10: What are common formats for industrial membrane units?
Folded cartridges for space saving Hollow fibre bundles (capillaries in tubes)
68
Q11: What is the difference between dead-end and cross-flow filtration?
Dead-end: Flow perpendicular to membrane, prone to clogging Cross-flow: Flow parallel to membrane, delays clogging
69
Q12: What are the advantages of ultrafiltration?
A: High recovery (up to 99%), fast processing, and low operational costs.
70
Q1: What is the principle behind precipitation in protein concentration?
A: Reducing biomolecule solubility using salts or organic solvents to induce aggregation and settling.
71
Q2: Why is precipitation commonly used in industry?
A: It’s simple, cost-effective, and widely applied for both proteins and polysaccharides—especially for bulk enzymes.
72
Q3: What is the most common salt used for protein precipitation?
A: Ammonium sulphate.
73
Q4: Why is ammonium sulphate preferred?
It’s inexpensive and soluble Stabilizes many proteins (NH₄⁺ ion) Doesn’t affect pH Effective at lower concentrations than other salts
74
Q5: What must be done after precipitation with ammonium sulphate?
A: The salt must be removed using dialysis, diafiltration, or gel filtration.
75
Q6: What protein concentration is recommended before precipitation?
A: Greater than 0.5 mg/mL.
76
Q7: How do organic solvents cause protein precipitation?
A: They lower the solution’s dielectric constant, enhancing electrostatic interactions and reducing solubility.
77
Q8: What is the dielectric constant and why does it matter?
A: It measures a solvent’s polarity; a lower constant (like ethanol or acetone) makes water less able to shield charges, promoting precipitation.
78
Q9: Why is water a good solvent for biomolecules?
A: Its high dielectric constant (80.8) shields electrostatic interactions and helps maintain protein solubility.
79
Q10: What are some common solvents used for precipitation?
A: Ethanol and acetone (frequently used for sterols and blood fractionation, respectively).
80
Q11: What are risks of using organic solvents in protein processing?
A: Risk of denaturation, especially for enzymes.
81
Q12: What is the typical solvent concentration needed to precipitate proteins?
A: Between 5% and 60%.
82
Q13: What other agents are used in small-scale precipitation?
Polyethylene glycol (PEG): reduces available water Polyacrylic acid: forms charge-based complexes
83
Q1: What is liquid-liquid extraction used for?
A: To transfer a solute (biomolecule) from one liquid phase to another in which it is more soluble.
84
Q2: What determines extraction efficiency?
A: The partition coefficient: [Concentration in extract phase]/[Concentration in raffinate phase]
85
Q3: What is a common application of liquid-liquid extraction in biotech?
A: Extraction of penicillin.
86
Q4: How is the solvent removed after extraction?
A: Using an evaporator under low pressure (vacuum), which reduces the solvent's boiling point.
87
Q5: Why is solvent use limited in processing biomolecules?
A: Organic solvents can denature sensitive macromolecules, so they are mainly used with more stable compounds like antibiotics.
88
Q6: When is evaporation used in DSP?
A: For large-scale water removal from aqueous biomolecule solutions.
89
Q7: What is typically used to heat the liquid in evaporation?
A: Steam, often in a vacuum to lower boiling points and protect product integrity.
90
Q8: Describe how a falling film evaporator works.
A: The liquid flows down heated vertical tubes as a thin film, promoting rapid evaporation; residence time is usually <30 seconds.
91
Q9: Why is vacuum evaporation preferred for biomolecules?
A: It reduces thermal damage by allowing evaporation at lower temperatures.
92
Q10: What is the purpose of using adsorbents in DSP?
A: To capture biomolecules on solid surfaces for concentration or purification.
93
Q11: What are common adsorbents used?
A: Activated charcoal and ion exchange resins.
94
Q12: How are ion exchange resins used outside of column formats?
A: Mixed as a slurry with the fluid, allowed to bind, then filtered or transferred to a column for elution.
95
Q13: What are the benefits of ion exchange resins?
High binding capacity Molecular sieving (aids purification) Resistant to harsh cleaning Cost-effective

BIOL3002 Biotechnology 2: Downstream Processing (4 decks)

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