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Pharmaceutical Biotech > CA1 > Flashcards

CA1 Flashcards

1
Q

What are biopharmaceuticals

A
  • pharm drug product produced through biotech in living system
  • diverse category eg. immunotherapies, blood components
  • large complex molecules composed of sugar, proteins, nucleic acid, complex combi
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2
Q

Biopharmaceuticals found in

A

clinical devices & diagnostics
eg. antigen rapid test
antibody test: distinguish current & past infection
molecular test (PCR): current infection

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3
Q

Small molecules vs biopharmaceuticals (size, MW)

A

typically <1000Da
vs
larger, typically in kDa

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4
Q

Small molecules vs biopharmaceuticals (complexity)

A

simple & well defined
vs
more complex

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5
Q

Small molecules vs biopharmaceuticals (production)

A

chemical process, controlled & predictable reactions
vs
derived from living sources, req bact/host cells, more costly, extensive quality control

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6
Q

Small molecules vs biopharmaceuticals (characterisation)

A

standardized, final structure easily verified, contaminants quantifiable
vs
not easily characterised, refined to high deg of purity, batch to batch variation

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7
Q

Advantages of biopharmaceuticals

A
  1. SPECIFICITY: high specificity (larger size & greater extent of interaction)
  2. TOXICITY: lower toxicity
  3. METABOLISM: more predictably broken down by hydrolysis
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8
Q

Limitations of biopharmaceuticals

A
  1. STABILITY: Heat sensitive, limited shelf life
  2. IMMUNOGENICITY: potentially immunogenic
  3. ABSORPTION: barriers in GIT, need invasive delivery
  4. UPTAKE: limited ability to cross cell membrane
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9
Q

Biopharmaceutical modalities

A

Proteins, antibodies, nucleic acids & engineered cells

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10
Q

Biotechnology milestones: emergence

A

to be consistently, safely & cost effectively applied need the basic principles of recombinant DNA, genetic engineering, bioprocessing technologies

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11
Q

Limitations & driving forces of biopharmaceutics that lead to biotechnology development

A
  1. Identification of biomolecules
  2. Biomolecule produced naturally in exceeding low qty (low production by native sources) –> limited therapeutic/medical applications
  3. Chemical synthesis not useful for large proteins
  4. Product safety - biomolecule isolated from native sources may be immunogenic/contaminated
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12
Q

Enabling biotech: The Central Dogma

A

DNA transcribed –> RNA translated –> Protein –> Cell

  1. Genetic Eng
  2. Produce recombinant macromolecules
  3. Engineering & scalable culture of host cells
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13
Q

Genetic engineering

A

Reverse dogma –> production of proteins with known amino acid sequence

  • restriction enzymes (cleave DNA)
  • DNA polymerases (sequence & amplify DNA)
  • manipulate & propagate DNA using bacterial plasmids
  • PCR Tech
  • Sequencing
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14
Q

Emergence of biopharm: Monoclonal antibodies

A
  1. Hybridoma Tech (large-scale production of monoclonal antibodies)
  2. Phage display & recombinant antibody engineering (in vitro selection of human mAbs of any specificity & affinity)
  3. Cell engineering (engineered host cell systems eg. CHO cells for industrial manufacturing manufacturing of recombi protein facilitated by genomic engineering tools eg. CRISPR)
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15
Q

Genetic engineering propel rapid advancement of biotech

A
  1. Human recombi protein can be produced easily in large qty
  2. DNA sequence optimized (mutagenesis) –> enhance protein yield
  3. Protein modified (mutagenesis) to improve molecular characteristics. eg. PK
  4. Availability of highly specific antibodies –> assist purification & characterization of proteins
  5. Cells modified to improve function/create new functions
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16
Q

Gene Cloning Process

A
  1. PCR to amplify gene of interest
  2. Digestion of vector & gene of interest
  3. Ligation
  4. Transformation into host cells (insertion of recombinant DNA)
  5. Selection of required rDNA & propagate cells
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17
Q

PCR

A
  • Cloning of DNA outside in cell-free environment using thermostable DNA polymerases (<2h)
  • Pure samples of individual gene separated from a mixture of genes (based on specificity of primers)
  • Performed in thermocycler
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18
Q

PCR process

A
  1. Double stranded DNA
  2. Denature (95degC) to separate DNA strands
  3. Annealing of site specific primers (50-60degC)
  4. Elongation (72degC) - incorporate dNTPs to extend DNA
    Cycle repeated 30x
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19
Q

Limitations of PCR

A
  1. Sequences of primer annealing sites must be known (site directed mutations)
  2. Length of DNA sequence that can be copied by PCR (up to 40kb need to deal with specialised techniques)
  3. Infidelity –> no 3’ 5’ exonuclease (proofreading function), can lead to error (base misincorporation) ; can use alternative heat-stable DNA polymerases with 3’ 5’ exonuclease activity eg. Pfu DNA pol
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20
Q

Applications of PCR

A
  1. Amplify DNA fragment for gene cloning (genetic eng)
  2. Diagnostic applications
    - PCR amplification of mutant alleles –> determine if person is carrier of genetic disease
    - Early detection of disease/genetic abnormalities
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21
Q

Other tools & techniques for DNA work

A
  1. DNA purification from cells
  2. Gel electrophoresis
  3. Restriction enzymes for DNA cloning work
  4. DNA sequencing
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22
Q

DNA Sequencing (Sanger sequencing) –> chain termination method determine nucleotide sequence of DNA

A
  1. gene of interest cloned into vectors (dsDNA converted to single stranded by denaturation –> thermal cycle sequencing by DNA polymerase using 1 primer)
  2. enzymatic DNA polymerase synthesis of 2nd strand of DNA (in 5’ to 3’ direction), complementary to existing template
  3. chain terminates with fluorescent dideoxynucleotides (low conc & random incorporation) 3’ OH absent
  4. detection by capillary electrophoresis (separate by size)
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23
Q

Sanger-Coulson (chain termination) sequencing

A

position where -OH of dNTP replaced by -H; phosphate group cannot be added to elongate the chain
no 3’ -OH group

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24
Q

Automated (Dye-terminator DNA sequencing)

A

Fluorescent probes used for automated sequencing (diff fluorescent labels attached to each type of dideoxynucleotide)
- separate according to size, each fragment differ by 1 nucleotide

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25
Q

Next Generation Sequencing

A
  1. Next Generation Sequencing (sequence many genes at the same time)
  2. Nanopore Sequencing (longer DNA/RNA reads)
  3. Single cell sequencng
    eg. B-cell receptors & T-cell receptors
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26
Q

Pharm application for sequencing

A

Identify causal genes & design drug targets

- Personalized medicine –> design more specific drugs, predict drug-genes interaction to prevent adverse drug reactions

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27
Q

Protein drugs

A
  • 3-D shape determines biological function of protein
  • Recombinant protein (genetically engineered version of naturally occurring human proteins)
  • Replace a protein that is abnormal/deficient/augment the body’s supply of beneficial protein
  • Advantage: high specificity, activity
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28
Q

Challenges of protein drugs

A

Immunogenicity, stability, drug delivery (poor oral bioavailability, rely on IV)

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29
Q

Challenges of protein drugs (immunogenicity)

A
  • Foreign proteins –> immunogenic response
  • Loss of efficacy (development of antibodies in host body)
  • Anaphylactic shock in severe rxn
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30
Q

Challenges of protein drugs (protein stability)

A
  • Proteins subjected to a wide range of influences –> loss of biological activity
  • Destroy protein’s biological activity by inducing denaturation –> loss of proper 3D conformation

Loss of biological activity can occur during:

  1. protein recovery from its source (extraction)
  2. protein purification
  3. post-protein purification (storage)
  4. storage for any length of time (proteolysis due to enzymes associated with bacterial contamination + degradation –> store in freeze-dried)
  • biological assays can reveal potency of product
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31
Q

Factors affecting protein stability

A
  1. Physical - aggregation

2. Chemical - deamidation, oxidation, proteolysis, disulfide exchange

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32
Q

Protein aggregation

A

Native (N) <=> Unfolded/Denatured (U) => Aggregated

Physical stability expressed as diff in free energy (change in G) between N & U

  • Unfolding: reversible/irreversible
  • Subsequent aggregation of denatured molecules: irreversible denaturation
  • Aggregated protein: may have altered activity; arouses immunogenicity
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33
Q

Mechanisms of Protein Aggregation

A
  • Due to intermolecular association of partially denatured protein chains
  • Hydrophobic force is major force for folding & aggregation

Factors

  1. Temperature** (slow unfolding at elevated temp)
  2. pH
  3. Ionic strength (higher salt conc, higher ionic strength higher stability but beyond optimal can denature)
  4. Vortexing
  5. Chemical modification of proteins
34
Q

Factors affecting protein stability

A
  1. Temperature** (increased temp promotes protein unfolding by disrupting non-covalent forces –> denatured proteins aggregate –> irreversible denaturation)
  2. pH (disruption of distribution of ionic attractive & repulsive forces)
    - also affect protein’s chemical stability: hydrolysis of Asp residues, deamidation of Asn & Gln
  3. Adsorption
    - proteins can be adsorbed to many surfaces –> change in secondary & tertiary structure
    - loss of proteins/destabilization of proteins
  4. Shaking & shearing
    - agitating incorporates air into solution –> unfolding of protein to minimize exposure of hydrophobic residues to air –> partial/complete denaturation
  5. Non-aqueous solvents
    - protein hydration shell disrupted
    - protein hydrophobic core exposed as polarity of aqueous solvent decrease –> protein unfolds
  6. Repeated freeze-thaw
  7. Photodegradation
    - risk of protein aggregation upon exposure to light
    - tryptophan-side chain cleavage of Trp
35
Q

Chemical Instabilities of proteins

A

Many chemical rxn
1. Deamidation** (pH-dependent, influenced by secondary structure (gamma helical & beta turn stabilize Asn residues), Asn(N) & GlnQ)

  1. Oxidation (depend on position)
    - Catalyzed by transitional metal ions
    - Thiol groups of C & M most easily oxidizable
  2. Disulfide bond breakage & formation (free C residues oxidize easily to disulfide bond) –> promote protein aggregation
  3. Hydrolysis (acid&base)
    - Asp-Gly (D-G), Asp-Pro (D-P) particularly labile
    - carboxyl group of Asp residue acts as catalyst
  • position of AA determine chemical reactivity
  • may not always affect conformation/activity
36
Q

Stabilization & Formulation of protein pharm (liquid) strategies

A
  1. Substitution & Chemical Modification (internal changing of structural chracteristics)
    (A) AA substitution/modification (via site-directed mutagenesis)
    - protein analogues (Cys replaced by Ser)
    - replacement of deamidation Asn sites
    (B) Intro of disulfide bond
    - stabilize folded form of proteins
    (C) PEGylation
    - chemical attachment of PEG
    - increase circulation time in blood, better safety profile
    (D) Acylation
    - chemical attachment of fatty acids to residues on protein surface
    - increase circulation time in blood eg. insulin detemir
  2. Changing properties of solvent, additives (external)
    - stabilizers
    - solubility enhancers
    - anti-adsorption & anti-aggregation agents
    - buffer components
    - preservatives & anti-oxidants
37
Q

Challenges of protein drugs - oral bioavailability

A

Proteins digested & broken down by enzymes in GIT –> unable to pass through cell barrier

38
Q

Peptide drugs

A

<40 AA residues

  • natural peptide analogs
  • agonist (activate receptors)/antagonist (disrupt protein-protein interactions /inhibit receptors)
39
Q

Challenges of peptides drugs

A
  • Poor PK (short half life, degradation, high lvls of clearance)
  • Poor oral bioavail (low GI stability, poor membrane permeability)
  • Stapling, cyclization, glycosylation to improve PK & bioavail

Stapling: connecting Cys residues with disulfide bond –> prevent unfolding

Cyclization: 3D conformation

40
Q

Drugs where short circulatory half life of drug is preferred?
Option 1: GLP-1 (enhance glucose stimulation of insulin)
Option 2: Angiotensin (vasoconstriction)
Option 3: Vasopressin (antidiuretic)

A

Ans: Angiotensin

administered during septic shock –> increase bp transiently, short half life ensures HTN is avoided

41
Q

Macrocyclic peptides

A
  • Natural products/derivatives
  • De novo generated cyclic peptides
  • Large SA (increase interactions –> higher affinity & specificity), resistance to proteases & cross cell membrane (after modify)
  • Eg. Desmopressin (oral) –> cyclization lead to resistance to metabolism, hydrophobic nature enhance cellular absorption across gut
42
Q

Designing peptide drug

A
  1. Identify hits through genetically encoded platforms generating libs of cyclic peptides
    - Phage Display
    - mRNA Display –> in vitro selection of functional proteins (constructs in which a protein is covalently attached to the mRNA that encodes it) –> yields de novo & rare proteins
  2. Enable identification of potential leads against challenging protein targets (lack traditional pockets to which small molecules can bind)
43
Q

Process of mRNA display

A
  1. DNA library
  2. transcribe DNA to mRNA
  3. ligate mRNA to puromycin (jump into A side at stop codon & link to nascent growing peptide strand –> link mRNA to peptide)
  4. translate mRNA to protein
  5. reverse transcribe mRNA (short shelf life) to cDNA (strong stable)
  6. affinity chromatography (POI exposed to cDNA-protein fragment)
  7. wash away unbound mRNA-protein
  8. elute bound mRNA-protein
  9. reverse transcribe mRNA to cDNA –> PCR to identify sequence of mRNA + amplify
44
Q

Recombinant protein making/synthesis (bioprocessing)

A

Driving forces
- Natural sources rare, ex –> cannot meet with dd
thus Recombinant DNA tech allows cheaper, safer & abundant supply (depending on efficiency of purification steps)

  • Stringent FDA req (purity, biological activity)
  • Robust purification process
45
Q

Things needed to produce recombinant proteins

A

Proteins, serum, sugars, lipids, etc

Cell culture (temp, CO2, O2, humidity)

46
Q

Types of host cells used for biopharmaceutical production

A

Bacteria

  • insufficient folding of complex proteins of higher organisms - inclusion bodies
  • lack of post-translation modifications
  • endotoxins

Yeast

  • post-translational modifications differs from mammalian cells
  • problematic cell disruption (thicker cell wall, harder to disrupt)
  • protease degrade foreign proteins

Insect & mammalian

  • laborious construction of over-expressing strains
  • expensive media
  • low growth rates
  • difficult scale-up
  • *mammalian cells most physiologically relevant but costly

Transgenic plants & animals

  • long developmental cycles
  • contamination problems (animal viruses, prions)
47
Q

Expression system selection (choice of host cells to make recombinant proteins)

A

Depends on size & characteristics of proteins

  • Large proteins: Eukaryotic hosts
  • Small proteins: Prokaryotic hosts (E. coli)
  • If post-translational modification essential: Eukaryotic
  • If solubility & native folding crucial: Eukaryotic
  • High yields, low cost: Prokaryotic
48
Q

Host cells - Bacteria (E.coli)

A

eg. protein produced is insulin

Advantages:

  1. well characterized –> facilitates genetic manipulation
  2. high expression levels of recombinant protein (~30% of total cellular protein)
  3. grows rapidly on simple & inexpensive media

Disadvantages:

  1. Recombinant protein accumulates intracellularly
    - if synthesized in low levels, remain soluble in cells –> additional processing steps to purify from rest of host cell proteins
    - if synthesized rapidly & in high levels –> insoluble aggregates (inclusion bodies) –> easier to purify but need to undergo refolding to be active
  2. Unable to perform post-translational modifications eg. glycosylation
  3. Presence of lipopolysaccharides (LPS) on its surface –> pyrogens (can lead to immune rxn)
49
Q

CHO (Chinese Hamster Ovary) Cells

A
  • most common mammalian hosts (~70% of recombinant protein therapeutics produced in CHO cells)

preferred:

  1. Can adapt & grow in suspension culture –> large scale culture
  2. Less risk, few human viruses can propagate
  3. Can grow in serum-free & chemically defined media –> reproducibility between different batches
  4. Glycosylation of glycoproteins more human-like –> more compatible, bioactive
  5. Can be manipulated by genetic engineering techniques to produce higher yield of recombinant proteins
50
Q

Cell culture

A
  • change every 2 days generally
  • growth/dispersal of a collection of cells under lab cond
  • lab conditions: artificial environment - nutrient solution, suitable surface, ideal conditions (temp, humidity, gaseous atm)
51
Q

Applications of cell culture

A
  1. Toxicity testing of chemicals, new drugs, cosmetics etc
  2. Cancer research (cultured cancer cells to choose suitable drugs to destroy cancer)
  3. Virology (replication of viruses in vaccine production)
  4. Cell-based manufacturing –> 1. Large scale production of viruses for vaccine production, 2. Large scale production of cells that have been genetically engineered to produce proteins (recombinant protein & mAb production)
  5. Genetic engineering
  6. Gene therapy - cells removed from a patient lacking functional gene/replace damaged gene
52
Q

Advantages of cell culture

A
  1. Consistent & reproducible results (same batch of cells, homogenous popn)
  2. Effect of toxin on organ evaluated more accurately
  3. Less costly
53
Q

Disadvantages of cell culture

A
  1. Dependent on skill, knowledge & expertise
  2. Contamination of mammalian with bacterial, fungi etc
  3. After period of continuous growth, cell characteristics can change, accumulate mutations
  4. Cells can adapt to diff nutrients –> changes in intracellular enzymes
  5. Activities of intracellular enzyme may change if nutrient depletion/product accumulation in culture (produce byproduct as they consume nutrients)
54
Q

Nutrients in culture media

A

low MW
- sugar, AA, RNA & DNA precursors, lipids, bulk ions & trace metals, vitamins & enzyme co-factors

high MW

  • proteins (allow/promote cell growth & stimulate cells)
    1. hormones & growth factors (stimulate growth of cell lines)
    2. fibronectin, laminin, virtronectin (promote attachment of cells)
    3. insulin (promote glucose uptake)
    4. transferrin (iron transport into cells)
  • serum
    1. increase effectiveness of chemically defined media
    2. complex mixture w many functions eg. carrier proteins, hormones, protease inhibitor, nutrients
    3. eg. foetal bovine serum, newborn calf serum, donor calf serum, human serum
  • additives
55
Q

Nutrients in culture media (SERUM)

A

most common: Foetal Bovine Serum (FBS)
- contain high conc of growth stimulatory factors, lower conc of growth inhibitory factors

Problems:

  • Variability
  • High cost
  • Risk of animal-borne virus
  • Protein content needs to be removed during purification steps –> burden

FDA: to avoid all animal products, animal component-free media to be used

56
Q

Serum-free & animal component-free media

A 
1. Serum-free media
Growth factors & hormones may be needed
- growth & maintenance factors
- transport proteins
- other hormones
- cytokines eg. IL-6

Animal Component-free media

  1. Non-animal products may also be used
  2. Proteins from non-animal sources eg. recombinant insulin & other proteins from E.coli
57
Q

Cell culture (culture media)

A 
Basal medium (DMEM, IMDM, MEM etc)
- contains inorganic salts, AA, vitamins, pH buffer & indicator, glucose

Sufficient to support mammalian cell growth?
- req supplementation w serum, proteins and/or protein hydrolysates for mammalian cells to grow

58
Q

Cell development using CHO cells (Steps 0&1)

A

Step 0: Gene cloning
Step 1: Delivery of gene of interest (GOI) through vector into host cell’s nucleus –> site specific integration (CRISPR) / random integration of linearized vector

59
Q

Cell development using CHO cells (Step 2)

A

Step 2: Selection

  • Cells transfected with vector with GOI & DHFR enzyme
  • Cells cultured in medium lacking GHT & low levels of MTX
  • — Cells are CHO mutants lacking DHFR
  • — Cells without DHFR cannot grow in absence of GHT
  • — MTX inhibits DHFR –> select for cells expressing DHFR
60
Q

Cell development using CHO cells (Step 3&4)

A

Step 3: Recovery & expansion
- Surviving cells express protein of interest –> recovered –> varied protein productivity

Step 4: Amplification
- Surviving cells exposed to high conc of MTX –> increase selection pressure –> amplification of locus of DNA integration –> increase no. of DHFR + protein of interest

61
Q

Cell development using CHO cells (Step 5, 6, 7, 8)

A

Step 5: Screening
- Select individual clones with highest possible productivity & growth rates & best quality

Step 6 & 7: Expansion & Evaluation
- Chosen clones expanded, evaluate each clone in lab-scale bioreactors under industrial conditions

Step.8: Cell banking
- Single production cell line chosen & banked (master cell line/stock) as frozen vials

62
Q

Bioreactors for cell culture - adherent cells

A
  • Req surfaces for attachment, spread, growth
  • T-flasks
  • Roller bottles
  • Cell factories
  • Microcarriers (small solid/porous spheres for cell attachment & growth –> high surface to vol ratio) –> coated with +charged polymer –> cells attach through electrostatic interactions
  • Fluidized bed bioreactor
  • Packed bed bioreactor
63
Q

Bioreactors for cell culture - suspension cells

A
  • Preferred due to ease of propagation
  • Shake flasks (gentle rotational motion)
  • Spinner flasks (stirring motion)
  • Wave bioreactors
  • Other bioreactors
64
Q

Industrial Bioreactors

A

Stirred tank bioreactor

  • For microbial fermentation & animal cell cultivation
  • Autoclavable, capable of monitoring pH, nutrient conc
  • Can be scaled up
65
Q

Recombinant Protein Making/Synthesis

A

Gene cloning –> Transfect and select CHO cells producing high lvls of recombinant proteins –> Culture in bioreactors –> extract & purify proteins

66
Q

Protein Purification Process (Downstream Processing)

A
  • Recover a protein from its source (host cells)
    1. Extraction (Lysis)
    2. Enrichment/isolation of protein of interest (Purification)
    3. Removal of interfering/contaminating substances
  • Protein of interest expressed extracellularly (separation of cells from media, collect media) / intracellularly (cell harvesting followed by cell disruption)
67
Q

Cell disruption (cells)

A

Mammalian cells
- protective cell wall absent

Microbial cells

  • presence of microbial cell wall
  • bacteria (peptidoglycan); yeast (beta-glucan)

Plant/Animal Tissues
- homogenization by rotating blades

68
Q

Techniques for cell disruption

A
  • Homogenization –> high shear forces
  • ultrasonication
  • glass bead milling (glass beads 0.2-0.3mm agitated vigorously with cells in a suitable lysis buffer)
  • osmotic shock
  • repeated freezing & thawing
  • enzymatic lysis (lysozyme)
  • detergent-based lysis (use ionic detergents)
69
Q

Protein Purification Process 1 (Removal of whole cells & cell debris)

A
  1. Removal of whole cells & cell debris
    - Centrifugation (cells 300-5000g for 5-15 min ; cell debris 10,000g for 45 min, according to density, size)
    - Filtration
    a. depth filter –> decreasing pore size rating
    b. membrane filter –> 0.2-10 micrometre
    - Aqueous Two-Phase Partitioning –> cells & cell debris partition to the lower, more polar & denser phase; soluble proteins partition to the top, less polar & less dense phase –> polymer-salt system/polymer-polymer system
70
Q

Protein Purification Process 2 (Removal of Nucleic acid & lipids)

A
  1. Removal of Nuclei acid & lipids

Nucleic acid removal by:

  • Precipitation using cationic Polyethylenimine (bind to -ve DNA)
  • Treatment with nucleases

Lipid:

  • act as contaminants & interfere with subsequent purification steps (clog chromatographic columns)
  • removed by passing solution through glass wool/cloth of very fine mesh size
71
Q

Protein Purification Process 3 (Concentration of protein of interest)

A
  1. Precipitation
    a. addition of neutral salts: salting out –> low ionic strength salting in; high I salting out –> salts compete with proteins for water of hydration –> protein precipitation
    * ammonium sulfate most common –> high solubility, inexpensive, lack denaturing properties

b. pH adjustment
- —- pH > pI: protein negatively charged
- —- pH = pI: precipitation
- —- pH < pI: protein positively charged

c. addition of organic solvents (ethanol, isopropanol, acetone)
- —- progressively displace water from the protein surface –> disrupt hydration shell + protein precipitation
* precipitants need to be removed before further processing
* inefficient precipitation if initiail protein conc is low

  1. Ion-exchange chromatography
    - Negatively-charged proteins of interest: use Anion exchangers
    - Positively-charged proteins of interest: use Cationic exchangers
    - Proteins bound to exchanges eluted from ion exchange column by adding salt soln of high ionic strength
    - Ion exchangers: functional grps covalently linked to porous beads made form cross-linked dextran, agarose/cellulose
  2. Ultrafiltration
    - molecular mass 1-300 kDa
    - little adverse effects, high recovery rates (alot of proteins retained), rapid
72
Q

Protein Purification Process 4 (Purification of protein of interest)

A

Column chromatography: partition between 2 phases –> solid stationary phase (beads) & mobile phase (buffer)

  • size exclusion
  • ion exchange
  • hydrophobic interaction
  • affinity
  • immunoaffinity

separate according to:

  • MW/size
  • charge
  • hydrophobicity
  • affinity

Gel Filtration Chromatography
- based on size (MW & shape), in decreasing molecular size –> large proteins eluted faster

Ion Exchange Chromatography

  • based on reversible electrostatic attraction of charged protein to solid matrix of ion exchangers
  • cation exchange: matrix -ve charged
  • anion exchange: matrix +ve

Hydrophobic Interaction Chromatography

  • based on proteins’ surface hydrophobicity
  • protein samples applied to column in a buffer of high ionic strength –> proteins retained
  • elution of protein of interest: decrease hydrophobic interaction using buffer of lower ionic strength/lower polarity of buffer

Affinity Chromatography

  • specific & reversible binding of proteins of interest to affinity matrices
  • ligands covalently attach to inert support matrix; protein of interest bind to immobilized ligand
    a. General ligand approach –> elute protein of interest by changing buffer pH, ionic strength/polarity of buffer –> specific, selective but expensive & poor stability of ligands
    b. Specific ligand approach: immunoaffinity purification
  • involve conditions resulting in partial denaturation of bound protein eg. acidic pH ; milder conditions eg. increase in ionic strength of eluting buffer
73
Q

Protein Purification Process: Removal of contaminants

A

Potential contaminants:

  1. host-related: viruses, host-derived proteins, DNa
  2. product-related: AA substitution & deletion, etc
  3. process-related: grow medium components, purification reagents, metals & column materials
74
Q

Microbial Consideration

A
  1. Sterilization
  2. Viral decontamination (heat, irradiation, sonication, extreme pH)
  3. Pyrogen removal (dry heat/anion exchange)
  • microfilter: 0.2-10micrometre
  • ultrafiltration: 1-20nm
75
Q

Synthesis of recombinant proteins (SUMMARY)

A
  1. Cloning recombinant DNA into vector
  2. Developing CHO cell lines expressing high levels of recombinant protein
  3. Extract recombinant proteins from host cells (cell disruption)
  4. Removing cell debris, lipids, nucleic acids
  5. Concentrating proteins
  6. Purification of recombinant protein
  7. Removal of contaminants
76
Q

Casein

A
  • major soluble protein (80%) found in milk
  • mixture of phosphoproteins derived from casin family of genes
  • exists in milk as calcium caseinate (isoelectric pt 4.6)
  • Ca-caseinate + 2H+ –> Casein + Ca2+
77
Q

Effect of pH of solvent on protein solubility

A
  • proteins are least soluble when pH = pI
78
Q

alpha-chymotrypsin

A
  • proteolytic enzyme (endoprotease) acting in the digestive systems ( cleave internal peptide bonds)
  • selective for peptide bonds with aromatic/large hydrocarbon side chains
  • cleaving N-succinyl-L-phenylalanine p-nitroanilide (large hydrophobic amino acid) –> p-nitroaniline (yellow0 released
79
Q

salting in/salting out of proteins

A
  • at low ionic strength –> salting in (increase protein solubility) –> counterions help dissolve protein
  • at high ionic strength –> salting out (precipitation) –> salts compete with proteins for water of hydration –. proote protein-protein interactions between hydrophobic patches on surface of adjacent protein molecules –> protein precipitation
80
Q

effect of temp on protein

A
  • high temp breaks up non-covalent interactions in tertiary structure of protein –> unfolding of polypeptide chain
  • as temp increase, bonds within protein break –> polypeptide chains unfold & expose hydrophobic molecules which form new bonds –> change in texture
81
Q

effect of dielectric constant of solvent

A
  • dielectric constant: ease by which a dielectric medium may be polarized
  • organic solvents eg. alcohol, acetone reduce protein solubility
  • —– reduce dielectric constant of solvent
  • —– disruption of hydration shell (water attracted away from proteins, charged molecules exposed in non-polar solvent, increase electrostatic attraction of proteins)
82
Q

acid/base hydrolysis of proteins

A

acid hydrolysis:
- heat under reflux with 6M HCl for 24h, acid amino acid ion formed

alkali hydrolysis

  • solution of aq NaOH > 100 degC
  • alkali amino acid ion (salt) formed

Pharmaceutical Biotech (1 decks)

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