UNIVERSITY COLLEGE LONDON

EXAMINATION FOR INTERNAL STUDENTS

MODULE CODE

ASSESSMENT
PATTERN
MODULE NAME

BENG2011

BENG2011C

Bioprocess Recovery and Purification

DATE

24-May-11

TIME

14:30

TIME ALLOWED

3 Hours 0 Minutes

2010/11-BENG2011C-001-EXAM-35
2010 University College London

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Answer FOUR QUESTIONS. ANSWER AT LEAST ONE QUESTION FROM PART A

AND ONE FROM PART B. ALL questions carry a total of 25 MARS each, distributed as
shown [ ]. Only the FIRST FOUR ANSWERS will be marked.

PART A
1.

a)

b)

c)

A novel (3-lactam antibiotic (pK

4.6) is to be produced commercially by

fermentation using liquid-liquid extraction with organic solvents as the
primary recovery operation. Define the thermodynamic parameters that
can be used to quantify the extent and selectivity of antibiotic extraction.

[7]

In order to specify the operating conditions of the large scale process you
have been asked to design a laboratory scale study of factors influencing
extraction of the antibiotic. Describe the factors you would investigate and
outline typical results that might be obtained.

[9]

Your laboratory experiments have indicated that the equilibrium

distribution coefficient of the new compound under the chosen extraction

conditions is 20. The harvest concentration of the compound in the

fermentation broth is 40 g kg4 and the mass flow rate of the extracting
solvent is planned to be 7-fold less than that of the aqueous feed. If a
minimum of 98% w/w recovery of the antibiotic is aimed for calculate the
number of theoretical stages required. Clearly state any other assumptions
made.
[9]

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2.

A 10,000 L batch reactor is used to prepare a protein precipitate from a

clarified fermentation broth. The objective of the operation is, when
combined with a continuous-flow disc stack centrifuge, to provide a

clarified broth from which the precipitate has been removed and
discarded. This sequence acts as a preparative stage for chromatographic
purification of a therapeutic protein.
The reactor and centrifuge operating conditions are given below.

You are asked to prepare a report on the scope for increased throughput
by reduction in the residence time in the reactor during the precipitate
preparation stage.
As part of this report please prepare the following.
i)

A flowsheet of the process including all pumps and hold tanks. This
flowsheet should include at the start the hold tank for the clarified
fermentation broth and at the end the feed line to the first
chromatography stage.

ii)

[5]

An analysis of the 10,000 L reactor stirrer speed and mixing time in

terms of the characteristics which will determine the precipitate

properties.
iii)

iv)

[8]

A recommendation of the reduction of time used for ageing of the

precipitate in the reactor with full reasons for your recommendation.

[5]

A design of a 10 L reactor to test the recommendation at pilot scale.

[7]

For information
Reactor specifications
Baffled tank; height = diameter =2.33 m
Stirrer diameter =0.78 in, stirrer speed = 20 rpm
Reactor fill time for clarified broth and precipitating agent = 1 h
Reactor mixing time for precipitate ageing= 12 h
Time to empty reactor contents to centrifuge= 5 h
Time for reactor turnaround = 4 h
Reactor operated once per day; factory operates 24 h/day using a 3 shift
system.

Properties of precipitate suspension; density 1000 kg ni3 ,viscosity 0.005

N s m-2

Please explain all assumptions made and show details of all

calculations.
CONTINUED
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3.

Describe the principles and key considerations for immunoaffmity

a)

b)

chromatography, giving a basis to cost such an approach.

[15]

How are developments in antibody technology making immunoaffinity

chromatography more attractive for bioprocessing?

[10]

PART B
4.

a)

c)

Explain why it is that the rate of filtration falls over time during the
filtration of a biological feed being separated by a coarse filter. You
should amplify your answer with suitable relationships explaining
clearly all terms you use.

[7]

How does a filter aid help improve filtration rates? Detail the two modes
in which such aids are used.

[6]

Using the following data, determine what you believe to be the best
practical strategy for the recovery of this valuable excreted product. In
your analysis there is one major potential source of additional product

loss which is not accounted for. What is this and how might you
minimise that loss?

[12]

Fermentation time

Expressed product titre

(h)

(Wla

6
10
15

12

20
25
30
35
40

20
35
50
60
60
60

Filtrate volume collected

in 10 mins (L)
1.0
1.0
0.9
0.85
0.8

0.75
0.5
0.2
0.1
0.1
0.1

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5.
a)

b)

Two basic sets of equations are used to describe and to subsequently size
centrifuges for solids separation. What are they? Provide full details and
explain all terms. Are there any limits to their application? If so, state
these.

[8]

Given the following data, calculate the time taken for a yeast cell to
travel 0.3mm; the gap between the disks on a typical disk stack machine.

(NB you may need to make assumptions. If so, state them and justify
them). What 'g' force equivalent is being created?

[12]

Suspension is water-like
Diameter of centrifuge bowl; 0.3m
Speed of rotation = 5000rpm
c)

Comment on how you might achieve containment of a separation of a

potentially hazardous product from the fermentation liquors if a tubular
bowl design of machine were to be selected.

[5]

What are the 3 main process functions of expanded bed

chromatography? How is each achieved and what is the order of priority
of them? Justify your answers.

[6]

Using sketch diagrams demonstrate how you would use protein release
data to calculate the rate constant for a high pressure disruption step?
Why does the rate of release of specific enzymes differ from that of total
protein?

[7]

6.
a)

b)

c)

Explain the importance of the membrane rejection coefficient and

provide a simple mass balance equation to show how you would
estimate the specific rejection coefficient for a given protein if you had
filtrate and retentate samples to hand.

d)

[6]

When selecting a membrane module design it is crucial to consider a

range of economic and operational characteristics. Taking the example
of a hollow fibre cartridge, detail the pertinent characteristics of such a
design.

[5]

END OF PAPER

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