LEC 2 SHORT Qs

Question 1

Q1: What is the most common method used for biopharmaceutical purification?


Answer 1

A: High resolution chromatography.

Question 2

Q2: Where is purification carried out in industrial settings?

Answer 2

A: In purification suites — Grade C or D, depending on whether systems are open or closed.

Question 3

Q3: How many chromatography steps are typically used in a purification process?

Answer 3

A: 2–4 steps.

Question 4

Q4: What else does chromatography remove besides proteins?

Answer 4

A: Non-protein contaminants like salts.

Question 5

Q5: What are the two modes of mobile phase flow in chromatography?

Answer 5

Isocratic elution: Same buffer throughout
Gradient elution: Gradually changing buffer composition during the run

Question 6

Q6: How is protein elution monitored?

Answer 6

A: By absorbance at 280 nm; fractions are collected automatically.

Question 7

Q7: What is the size range for industrial chromatography columns?

Answer 7

Pre-clinical: 1–2.5 cm diameter
Clinical trial: 5–10 cm diameter
Production scale: 14–45 cm diameter

Question 8

Q8: What is the typical purity achieved by chromatography?

Answer 8

A: 98–99% (normal resolution); further polishing may be used for certain products like insulin.

Question 9

Q9: What are ideal properties of chromatography resin?

Answer 9

Inert (no non-specific binding)
Rigid (handles high flow pressure)
Chemically stable (withstands cleaning)
Bead-shaped and porous (good flow, high surface area)

Question 10

Q10: What are the main stages of a chromatography operation?

Answer 10

a. Column conditioning
b. Loading crude product
c. Protein elution
d. Column regeneration
e. Column cleaning
f. Column sanitization
g. Storage

Question 11

Q1: What is gel filtration chromatography based on?

Answer 11

A: Size and shape of biomolecules; larger molecules elute first because they can’t enter gel pores, while smaller ones are delayed.

Question 12

Q2: What is the stationary phase in gel filtration?

Answer 12

A: A gel with defined pore sizes (e.g. Sephadex, Sepharose, Sephacryl).

Question 13

Q3: Why must sample volumes be small in gel filtration?

Answer 13

A: Only 2–5% of the total bed volume is usable to maintain resolution; thus, samples must be concentrated.

Question 14

Q4: What is a common use of gel filtration in purification workflows?

Answer 14

A: As a final polishing step to separate monomers from aggregates and degradation products.

Question 15

Q5: Give examples of gel filtration media and their fractionation ranges.

Answer 15

Sephadex G-50: 1,500 – 30,000 Da
Sephadex G-100: 4,000 – 150,000 Da
Sephacryl S-200 HR: 5,000 – 250,000 Da
Ultrogel AcA 34: 20,000 – 400,000 Da
Bio-Gel P-300: 60,000 – 400,000 Da

Question 16

Q6: What is the principle of elution in gel filtration?

Answer 16

A: Molecules that cannot enter the gel pores move faster and elute earlier than smaller molecules that penetrate the pores.

Question 17

Q1: What is the principle behind ion exchange chromatography?

Answer 17

A: Separation based on charge differences between proteins at a given pH and the oppositely charged groups on the resin.

Question 19

Q2: What determines a protein’s charge in IEX?

Answer 19

A: Its isoelectric point (pI) and the pH of the running buffer.

Question 20

Q3: How does ion exchange chromatography work?

Answer 20

Proteins bind to the resin via electrostatic attraction.
Elution is achieved by changing buffer pH or ionic strength (e.g. adding salt) to disrupt these interactions.

Question 21

Q4: What is a cation exchanger?

Answer 21

A: A negatively charged resin that binds positively charged proteins (e.g. carboxymethyl cellulose, sulphonate).

Question 22

Q5: What is an anion exchanger?

Answer 22

A: A positively charged resin that binds negatively charged proteins (e.g. DEAE-Sephadex).

Question 23

Q6: What are examples of strong and weak ion exchangers?

Answer 23

Strong cation exchangers: Sulfopropyl, sulfoethyl (pKa 1.1–2.0)
Weak cation exchangers: Carboxylic groups (pKa 3.7–4.3)

Question 24

Q7: How is elution typically performed in IEX?

Answer 24

A: By gradually increasing salt concentration or adjusting pH to weaken binding and release the proteins.

Question 25

Q8: What is the dynamic binding capacity of cation exchangers for IgG?

Answer 25

A: Around 100 g/L.

Question 26

Q9: Why is IEX widely used in biotechnology?

Answer 26

A: It's highly effective, applicable to most proteins, offers high resolution, good binding capacity, and easy scalability.

Question 27

Q1: What is the basis of hydrophobic interaction chromatography (HIC)?

Answer 27

A: Separation based on a protein's surface hydrophobicity — hydrophobic amino acids interact with hydrophobic ligands on the resin.

Question 28

Q2: What are common ligands used in HIC resins?

Answer 28

A: Butyl, octyl, and phenyl groups attached to a solid gel matrix.

Question 29

Q3: When is HIC typically used in a purification scheme?

Answer 29

A: Early in the process, to achieve both concentration and partial purification from dilute solutions.

Question 30

Q4: How are conditions in HIC different from those in ion exchange chromatography?

Answer 30

A: They’re nearly opposite: HIC uses high concentrations of non-denaturing salts to enhance hydrophobic interactions during binding.

Question 31

Q5: What is commonly used in the buffer to promote protein binding in HIC?

Answer 31

A: Ammonium sulphate (a non-denaturing salt).

Question 32

Q6: How are proteins eluted in HIC?

Answer 32

By decreasing the salt concentration in the buffer Alternatively, by lowering the temperature or adding ethanol

Question 33

Q7: Why does high salt promote binding in HIC?

Answer 33

A: High salt removes water from protein surfaces, exposing hydrophobic regions and enhancing their interaction with the resin.

Question 34

Q8: Why is HIC useful in combination with other purification methods?

Answer 34

A: It’s orthogonal to techniques like ion exchange — meaning it separates proteins based on different properties, helping improve overall purification.

Question 35

Q1: What is the principle behind affinity chromatography?


Answer 35

A: Specific, high-affinity, non-covalent binding between a protein and a ligand (termed an "affinitin").

Question 36

Q2: What are common ligand–target interactions used in affinity chromatography?

Answer 36

Antigen–antibody Enzyme–substrate Lectin–carbohydrate Antibody–Staphylococcal Protein A (SpA)

Question 37

Q3: What is SpA and why is it important in purification?

Answer 37

A: Staphylococcal Protein A is derived from S. aureus and binds strongly to the Fc region of human IgG — widely used in monoclonal antibody (MAb) purification.

Question 38

Q4: What is dye-ligand affinity chromatography?

Answer 38

A: A pseudo-affinity method using dye ligands (e.g., Cibacron Blue) that mimic natural ligands like nucleotides.

Question 39

Q5: What proteins bind to Cibacron Blue?

Answer 39

A: Enzymes requiring NAD/NADP (e.g., dehydrogenases, kinases, some blood coagulation factors).

Question 40

Q6: What is Procion Red used for in affinity chromatography?

Answer 40

A: To purify NADP-dependent enzymes and carboxypeptidase G.

Question 41

Q7: What is the function of immobilized cyclodextrins in affinity chromatography?

Answer 41

A: Binding lipophilic molecules.

Question 42

Q8: What does lysine agarose bind?

Answer 42

A: Plasminogen and ribosomal RNA.

Question 43

Q9: What makes affinity chromatography highly efficient?

Answer 43

A: It can achieve high-fold purification in a single step.

Question 44

Q10: How are bound proteins eluted in affinity chromatography?

Answer 44

A: By altering buffer conditions (pH, ionic strength) or by introducing a competing ligand to displace the bound target.

Question 45

Q11: What is metal chelate affinity chromatography based on?

Answer 45

A: Coordination between metal ions (e.g. Ni²⁺, Cu²⁺, Zn²⁺, Fe³⁺) and electron-donating amino acid side chains, especially histidine.

Question 46

Q12: How is the metal held in place on the resin?

Answer 46

A: A chelating arm on the resin provides partial coordination sites; the protein provides the remaining ligands to form a stable complex.

Question 47

Q13: What influences metal chelate binding efficiency?

Answer 47

Type of metal ion Length of spacer arm Matrix properties pH, buffer composition, and ligand concentration

Question 48

Q14: What proteins are commonly purified using IMAC?

Answer 48

A: Gamma globulins, interferons, and recombinant His-tagged proteins.

Question 49

Q15: What is a His-tag and how does it work?

Answer 49

A: A stretch of 6+ histidine residues genetically engineered into a recombinant protein that binds tightly to metal ions (usually Ni²⁺) on the resin.

Question 50

Q16: What is typically used to elute His-tagged proteins?

Answer 50

A: Imidazole or free histidine (competes with the tag for binding sites).

Question 51

Q17: How is the His-tag removed after purification?

Answer 51

A: A protease cleavage site (e.g., thrombin) is placed next to the tag so it can be cleaved off post-purification.

Question 52

Q1: What is hydroxyapatite and why is it used in chromatography?

Answer 52

A: A microcrystalline form of calcium phosphate (Ca₅(PO₄)₃OH) that interacts with proteins and nucleic acids through multiple mechanisms, making it a mixed-mode resin.

Question 53

Q2: What types of interactions occur on hydroxyapatite surfaces?

Answer 53

Cation exchange: Amino groups in proteins bind phosphate groups Anion exchange (theoretical): Carboxyl groups or DNA phosphates bind calcium Metal chelation: Strong binding of phosphate groups to calcium

Question 54

Q3: Which interaction type dominates in protein separations with HTP?

Answer 54

A: Cation exchange and metal chelation — anion exchange plays a minimal role.

Question 55

Q4: How are proteins typically loaded onto hydroxyapatite columns?

Answer 55

A: In a dilute phosphate buffer at neutral pH.

Question 56

Q5: How is elution from hydroxyapatite columns performed?

Answer 56

A: Using a gradient of increasing phosphate concentration (same pH); NaCl has minimal effect.

Question 57

Q6: How do different protein types elute from HTP columns?

Answer 57

Basic proteins: Elute at low NaCl (~0.15 M) Acidic proteins: Require moderate phosphate concentrations; not displaced by high NaCl

Question 58

Q7: What types of biomolecules are typically separated using hydroxyapatite chromatography?

Answer 58

Chromatin Phosphoproteins DNA and RNA Monoclonal antibodies Aggregates, dimers, viral contaminants, host cell proteins, endotoxins, SpA

Question 59

Q8: What are the disadvantages of hydroxyapatite resins?

Answer 59

Mechanical instability Low reusability High cost