The document discusses measurement of the volumetric mass transfer coefficient (KLa), which indicates the rate of oxygen transfer in a bioreactor. It describes various methods to determine KLa values, including chemical and physical techniques like the sodium sulphite oxidation method. The document also covers factors that affect KLa, and how KLa values are used to scale bioreactors from laboratory to production scale.

1. Measurement of KLa
Presented by
SHINDE ASHOK SAKHARAM
UPSTREAM PROCESS DEVELOPMENT (ANIMAL CELL
CULTURE) 1

2.  The determination of the KLa value for fermentation is
important in order to maintain adequate transfer of oxygen in
a bioreactor, for laboratory scale use or when scaling up to a
larger process
 The volumetric mass transfer coefficient, (KLa) indicates the
rate of oxygen used for fermentation, taking into account all
oxygen-consuming variables in the bioreactor
 KLa values are used in scaling up from laboratory scale to
pilot scale or production scale bioreactors
2
INTRODUCTION OF KLa
F. Garcia-Ochoa, E. Gomez / Biotechnology Advances 27 (2009) 153–176

3.  The determination of the KLa of a fermenter is essential in
order
 To establish its aeration efficiency and
 To quantify the effects of operating variables on the provision
of oxygen
 The equations describing oxygen transfer are based on
dissolved oxygen concentration
 The solubility of oxygen is affected by dissolved solutes
therefore pure water and a fermentation medium saturated
with oxygen have different dissolved oxygen concentrations
3
Cont…
F. Garcia-Ochoa, E. Gomez / Biotechnology Advances 27 (2009) 153–176

4. I) CHEMICAL METHOD
a) Sodium sulphite oxidation
b) Carbon dioxide
c) Hydrazine oxidation
d) Catechol oxidation
e) Glucose oxidase
f) Kryptone absorption
4
Determination of KLa method
Biotechnol. Bioeng. 43: 1139–1145.

5. Determination of KLa method
II) PHYSICAL METHOD
A) Gassing-out techniques
a) Static gassing out method
b) Dynamic gassing out method
B) Oxygen-balance Technique
5Biotechnol. Bioeng. 43: 1139–1145.

6.  The oxygen-transfer rates is determined by the oxidation of
sodium sulphite solution
 This technique does not require the measurement of dissolved
oxygen concentrations
 Based on the rate of conversion of a 0.5 M solution of sodium
sulphite to sodium sulphate in the presence of a copper or
cobalt catalyst:
Na2S03 + 0.5 02 = Na2S04
6
SULPHITE OXIDATION TECHNIQUE

7.  Oxygen enters solution it is immediately consumed in the
oxidation of sulphite, so that the sulphite oxidation rate is
equivalent to the oxygen-transfer rate
 Since the dissolved oxygen concentration, is zero then the
KLa may then be calculated from the equation:
dCL / dt = OTR= KLa . C* (i)
KLa = OTR/ C*
 Where, OTR is the oxygen transfer rate
 The volumes of the thiosulphate titrations are plotted against
sample time and the oxygen transfer rate may be calculated
from the slope of the graph
7

8.  The estimation of the KLa of a fermentation system by gassing-out
techniques depends upon monitoring the increase in dissolved
oxygen concentration of a solution during aeration and agitation
 The oxygen transfer rate will decrease during the period of aeration
as CL approaches C* due to decline in the driving force (C* - CL )
 The oxygen transfer rate, at particular time, will be equal to the
slope of the tangent to the curve of values of dissolved oxygen
concentration against time of aeration, as shown in Fig.
8
GASSING-OUT TECHNIQUES

9. Fig. The increase in dissolved oxygen concentration of a solution over a
period of aeration. The oxygen transfer rate at time X is equal to the
slope of the tangent at point Y
9

10.  To monitor the increase in dissolved oxygen over an adequate
range it is necessary first to decrease the oxygen level to a
low value
 Two methods have been employed to achieve this lowering of
the dissolved oxygen concentration –
 The static method and
 The dynamic method
10
Cont…

11.  First described by Wise (1951)
 The oxygen concentration of the solution is lowered by gassing the
liquid out with nitrogen gas, so that the
 Solution is 'scrubbed' free of oxygen
 The deoxygenated liquid is then aerated and agitated and increase in
dissolved oxygen monitored using some dissolved oxygen probe
 The increase in dissolved oxygen concentration is given by –
dCL / dt = KLa (C*-CL) (ii)
 Taking logarithm after Integration of equation (ii) we have
ln (C*-CL) = - KLa.t
11
STATIC GASSING OUT METHOD
AIChE journal 43 (7), 1904-1908

12. A plot of the In (C* - CL) against time of aeration, the
slope of which equals -KLa.
12

13.  This method generally used for the live cells
OTR = dCL / dt = KLa (C*-CL) – xQO2
 Where,
 x is the concentration of biomass and
 QO2 is the specific respiration rate (mmoles of oxygen g-L
biomass h- L)
 The term xQO2 is given by the slope of the line AB in Fig -1
13
DYNAMIC GASSING OUT METHOD
AIChE journal 43 (7), 1904-1908

14. Fig.1. Dynamic gassing out for the determination of KLa values.
Aeration was terminated at point A and recommenced at
point B.
14

15. • Equation (iii) may be rearranged as:
CL = -1/KLa{(dCL / dt)+ xQO2}+C* ----------(iv)
• Now from equation (iv), a plot of CL versus dCL/dt +
xQO2 will yield a straight line, the slope of which will
equal -1/KLa, as shown in Fig. 2
15

16. Fig. 2 The dynamic method for determination of KLa values
16

17. Fig. 3. The occurrence of oxygen limitation during the dynamic
gassing out of a fermentation
17

18. ADVANTAGES
 The dynamic gassing-out method has the advantage over the
previous methods of determining the KLa during an actual
fermentation and may be used to determine KLa values at different
stages in the process
 The technique is also rapid and only requires the use of a dissolved-
oxygen probe, of the membrane type
18

19. LIMITATIONS
 A major limitation in the operation of the technique is the range
over which the increase in dissolved oxygen concentration may be
measured
 It may be difficult to apply the technique during a fermentation
which has an oxygen demand close to the supply capacity of the
fermenter
 Both the dynamic and static methods are also unsuitable for
measuring KLa values in viscous systems
19

20. OXYGEN-BALANCE TECHNIQUE
 Use to measure KLa during fermentation process
 The amount of oxygen transferred is determined, directly into
solution in a set time interval
 The KLa may be determined, provided that CL and C* are
known, from equation (i)
dCL / dt = KLa (C*- CL)
Or OTR = KLa (C*- CL)
Or KLa = OTR/(C*- CL)
20

21.  The oxygen-balance technique appears to be the simplest
method for the assessment of KLa and
 Has the advantage of measuring aeration efficiency during a
fermentation
21

22. FACTORS AFFECTING KLa VALUES IN
FERMENTATION VESSELS
 A number of factors have been demonstrated to affect the KLa
value. Such factors include
 The air-flow rate employed in vessels
 The degree of agitation inside vessels
 The rheological properties of the culture broth and
 The presence of antifoam agents
22

23. PROCESS SCALE-UP
23

24. WHAT IS SCALE-UP?
 To take a manufacturing process from the laboratory scale to a
desired large scale at which it is commercially feasible
24

25. PURPOSE OF SCALE UP
 Meeting increasing drug demand
 Reduce cost of goods
AIM OF SCALE UP
 Similar/Identical product quality and process behaviour compare to
Lab scale process
25
PURPOSE AND AIM

26. A) Volume Independent parameter
a) pH
b) Temperature
c) Dissolved oxygen
d) Seeding density
e) Feeding schedule
26
PARAMETERS FOR SCALE UP

27. A) Volume dependent parameter
a) Mass transfer coefficient (KLa)
b) Agitation Power Number (P)
c) Impeller Tip Speed
d) Flow rate of Gases
e) Mixing time/circulation time
27
PARAMETERS FOR SCALE UP

28. HOW TO DO SCALE UP???
28

29. AGITATION PARAMETERS
29

30. GASSING PARAMETERS
30

31. 31