lecture 1

Question 1

what do sulphonamides do?

Answer 1

stops growth of gram positive bacteria. by mimicking a precursor in folate biosynthesis and stops bacterial growth. used to treat streptococcus

Question 2

Consequence of sulphonamides

Answer 2

1937 107 people died from poisoning y having traces of diethylene glycol in sulphonamides

Question 3

penicillin what does it do and how it was manufactured?

Answer 3

mould that suppressed bacterial growth, Chain and Florey discovered penillin therapeutic properties and discovered how to manufacture and purify penicillin.

Question 4

deep tank fermentation penicillin

Answer 4

requires glucose, lactose, corn steep liquor, sodium nitrate, calcium carbonate, phenylacetate. Penicillin needs air to grow, therefore needs to be areated. however bubbling sterile air caused severe foaming, therefore antifoam is used to alter the surface tension and pop the bubble.

Question 5

corn steep liquor

Answer 5

corn steep liquor is a waste product of the corn wet milling process, clean corn is soaked in water containing lactic acid and sulphur dioxide for 40 hours and then filtered and concentrated using evaporators

Question 6

fermentation before 1940

Answer 6

raw material was corn, milled and the germ and bran extracted before adding to hot water. corn mash is inoculated and left for 2 days. continuous distillation resulted in 50% water, 30% butanol and 15% acetone and 5% ethanol. this is transferred to a kettle to produce pure butanol, acetone and ethanol

Question 7

reactor design- oxygen transfer

Answer 7

sparger- gas into liquid to allow for better dissolving, impeller- cuts through water and bubbles will be broken. Rushton turbine is a high shear impeller and breaks air down to a smaller size. Baffles- obstructs water and directs flow

Question 8

semi synthetic penicillin

Answer 8

Introduction of the penicillinase-stable semisynthetic penicillins, such as methicillin and cloxacillin, to which the most of these benzylpenicillin-resistant strains are sensitive.Broad-spectrum semisynthetic β-lactam drugs, such as ampicillin made possible the treatment of infections caused by certain Gram-negative organisms which were not previously sensitive to penicillin therapy.

Question 9

statins

Answer 9

interfered with cholesterol, highest selling category of pharmaceuticals

Question 10

penicillium liquid cultures

Answer 10

Shear sensitive particles in non-Newtonian liquids with gas bubbles

Question 11

Penicillin can be purified using which unit operations

Answer 11

Crystialisation, activated carbon removal of pigments and adsorption on to the ion exchange resin

Question 12

Cephamycin B was washed off the ion exchange resin using

Answer 12

A water salt mixture

Question 13

Which unit operation is useful for recombinant proteins expressed in E. coli but is useless for proteins secreted by mammalian cells?

Answer 13

homogenisation

Question 14

Which unit operation is useful for recombinant proteins expressed in E. coli but is useless for proteins secreted by mammalian cells?

Answer 14

Hydrophobicity

Question 15

What is a theoretical plate

Answer 15

A measure of the shape of a single peak eluting off a chromatography column

Question 16

tangential flow filtration caking and gel formation can be reduce by

Answer 16

introducing turbulence

Question 17

Microfiltration of a recombinant protein using a pore size between 0.1 and 10 μm can be used to achieve

Answer 17

Removal of cells and cell debris

Question 18

The Installation Qualification (IQ) is the documented proof that facilities and equipment have been delivered and installed in accordance with the requirements and statutory safety regulations stipulated in the design qualification. The IQ may include which of the following?

Answer 18

Engineering drawing
Material specification and test results
Software code

Question 19

The flux across a filter membrane is proportional

Answer 19

Pressure differential

Question 20

In tangential flow filtration of a recombinant protein, if you keep the volume constant and replace filtrate with a buffer of choice

Answer 20

Alter the buffer composition around the protein

Question 21

. Live attenuated viral vaccines can produced in which systems

Answer 21

hen eggs and mammalians cells

Question 22

Vaccines against human papillomavirus have been commercially manufactured using which of the following

Answer 22

Insect cells

Question 23

The chaperone GroEL helps in production of VLP-based vaccines by which of the following

Answer 23

Helping fold the capsomere intermediate

Question 24

The 1918 Influenza Pandemic killed approximately how many people

Answer 24

10 million

Question 25

Between 1914-1918 the conflict associated with the first world war killed approximately how many military personnel

Answer 25

10 million

Question 26

Vaccines made using virus-like particles have which of the following advantages

Answer 26

Are not infectious

Question 27

The primary structure of a protein is important as it determines the

Answer 27

tertiary structure the molecule adopts

Question 28

The sum of all genes and intergenic DNA on all the different chromosomes of a cell is referred to the genome. Which statement is correct?

Answer 28

Genomes of differing size can have similar numbers of protein coding genes.

Question 29

Eukaryotes such as mammalian cells

Answer 29

Transcription takes place in the nucleus Translational takes place in the cytoplasm mRNA is transported from the nucleus to the cytoplams

Question 30

protein post translational modifications increase the functional diversity of proteins through

Answer 30

Covalent addition of chemical groups proteolytic cleavage cleavage of regulatory subunits

Question 31

protein post translational modifications

Answer 31

Post translational modifications are most often carried out by enzymes

Question 32

post translational modification glycosylation

Answer 32

addition of sugars to proteins, that ranges from simple monoscharrides to to highly complex polyscharrides. eg monoclonal antibodies

Question 33

genetic engineering

Answer 33

1) isolate plasmid from bacterium 2) isolate gene of interest into plasmid 3) insert gene of interest into plasmid 4) bacterial cell takes up plasmid containing gene of interest through process called transformation 5) bacterial cell divides along with the plasmid and forms a clone cells/ genetically identical cells

Question 34

plasmids are important

Answer 34

1. they are easy to work with 2. self replicating 3. stable 4. functional in many species and can be useful for a wide set of applications

Question 35

what are chromosomes

Answer 35

genetic material commonly packaged into structures

Question 36

genome

Answer 36

the sum of the gene and intergentic DNA on all the different chromosomes

Question 37

protocol for purifying insulin

Answer 37

1. fermentation 2. disrupt cells (homoginisation) 3. wash inclusion bodies (centrifugation) 4. dissolve inclusion bodies 5. refold protiens 6. purify proinsulin (crystalisation) 7. cleave from the c-peptide (trypsin) 8. purify the insulin

Question 38

protocol for purifying growth hormones

Answer 38

1. fermentation 2. disrupt cells (homogenisation) 3. polyethyleneimine precepitation of nucleic acids and lipid 4. ammonia sulphate preceptiation 5. purify the growth hormone

Question 39

reactor design- inlet and outlet filters

Answer 39

air filters needs to have an air flux sufficient to supply oxygen for o2 transfer. the filters need to be hydrophobic so they dont get wet and reduce flux

Question 40

air flow from a sparger below a rushton operating at different speeds

Answer 40

cause high shear, and breaks air bubbles down to a smaller size which in turn improves o2 transfer. this is incompatible for microorganisms or mamallian cells, plant cells and insects that are sensitive to shear. yeast and bacteria and fungi have a high resistance to damage

Question 41

controlled parameters in a bioreactor

Answer 41

temp dissolved o2 foam level ph

Question 42

reactor design considerations

Answer 42

o2 transfer heat transfer-cooling jacket,heat exchanger aseptic operations- aseptic fittings,CIP attachments or double mechanical seals on the motor

Question 43

refolding in a reactor comapred to a cell

Answer 43

refolding within a cell is different from folding in a reactor and it takes place while it is attached to the ribosome. has a slow last stage as it needs to be protected from interference by chaperones

Question 44

e coli process

Answer 44

key points 1. high yield fermentation 2. moderate refold yield 3. low refold concentration 4. poor overall purification yield 5. complex process 6. produces less material for double the cost

Question 45

CHO process

Answer 45

1. low yield formation 2. high cost of fermentation 3. good yield purification 4. simple process 5. expensive

Question 46

microbial cells are used to make

Answer 46

insulin, growth hormone, factor VII, G-CSF

Question 47

mammalian cells used to make

Answer 47

monoclonal antibodies, tissue plasminogen activator

Question 48

isoelectirc point

Answer 48

pH of the protein has no net charge,

Question 49

ion exchange chromatography

Answer 49

separates protiens according to charge, variables that can be altered-> pH solution, strength of ligand

Question 50

Advantages of ion exchange chromatography

Answer 50

relatively cheap

Question 51

Disadvantages for ion exchange

Answer 51

poor separation as it cannot separate proteins with similar charges

Question 52

hydrophobic interaction and reversed phase

Answer 52

hydrophobic->salts Reversed-> elute solvent with with acetontrile or isopropanol. and binding is entropy driven due to the energy level of the water structure around the hydrophobic zones of the protein

Question 53

advantages of hydrophobic interaction

Answer 53

cheap

Question 54

disadvantages of hydrophobic interaction

Answer 54

poor seperation if used as a simple on off process

Question 55

variables that can be altered with hydrophobic interaction

Answer 55

compostion of the loading, washing and elution solutions. strength of the ligand

Question 56

affinity chromatography

Answer 56

interactions with specific molecules, seperates according to specific binding of a ligand to the protein surface

Question 57

variables you can alter with affinity chromatography

Answer 57

composition of loading, washing and elution solutions

Question 58

advantages of affinity chromatography

Answer 58

high resolution

Question 59

disadvantages of affinity chromatography

Answer 59

expenisive

Question 60

Size exclusion chromatography

Answer 60

used in buffer exchange of purified recombinant protiens, resolution- measure of overlap in chromatography. large materials come out first

Question 61

advantages of size exclusion chromatography

Answer 61

desalting or buffer exchange, removal of aggregates, seperation of macromolecules, mild process

Question 62

Disadvantges of size exclusion chromatography

Answer 62

vulnerable to cracking, channelling, caking unsutiable for crude starting materials dilutes products limits on scale up

Question 63

rotary drum vacuum filter

Answer 63

preforated drum is covered by a cloth precoat of diatomaceous earth Drum is immersed in slurry material is drawn through the cloth and out via a rotary valve liquid drawn through the cloth and out via a rotary valve solids removed by a roller or scraper

Question 64

advantages of a rotary drum vacuum filter

Answer 64

can process large volume of liquids, cheap to operate

Question 65

disadvantages of a rotary drum vacuum filter

Answer 65

difficult to operate aseptically

Question 66

tangential flow filteration

Answer 66

doesnt allow cake to build up, microfilteration removes cells debris and inclusion bodies. ultrafilteration removes macromolecules. there is an increased flow rate along the membrane, introduction of turbulance imporves perfromance of the membrane

Question 67

advantages of tangential flow

Answer 67

``` effective method of seperation relatively low shear suitable for most proteins and microorgainisms amenable to CIP and steam in place linear scale up simple to use ```

Question 68

disadvantages of tangential flow

Answer 68

gel layer formation reduces flux and can cause aggregation of proteins membranes relatively expensive to replace

Question 69

validation

Answer 69

is the process of establishing documentary evidence demonstrating that a procedure, process or activity carried out in production or testing maintains the desired level of compliance at all stages

Question 70

installation qualification

Answer 70

documented proof that facilities and equipment have been delivered and installed in accordance with the requirements.

Question 71

design qualification

Answer 71

defines the functional and operational specifications of the facility, process and ancillary equipment

Question 72

operational qualification

Answer 72

test process that evaluates the correct functioning of a facility or appliance

Question 73

Process qualitifcation

Answer 73

in starting all manufacturing equipment

Question 74

penicillin manufacturing process

Answer 74

Whole broth extraction is usually performed at acidic pH by most manufacturers and has resulted in a 2–5% improvement in overall extraction efficiency by the elimination of the rotary vacuum filtration step. Solvent extraction of chilled acidified broth is carried out with amyl, butyl or isobutyl acetate. Multiple back-extractions into buffer and solvent at varying pH using countercurrent contactors has led to considerable penicillin concentration in the early recovery stages of the purification process. Pigments and other broth impurities are removed by the use of activated charcoal. The penicillin is crystallized upon the addition of potassium acetate and is isolated as a crystalline potassium salt. Additional carbon treatments and solvent washes results in a highly purified final product