BioPharm Enewsletter Case Study
THE FEASIBILITY OF TANGENTIAL FLOW FILTRATION FOR CELL
LYSATE CLARIFICATION
INTRODUCTION
Tangential flow filtration (TFF) is an efficient and unavoidable downstream
processing (DSP) method for the clarification and purification of biologics.
Membrane filtration is a solid – liquid separation technique that has been
widely used in the biotech industry for many years. Depending on
membrane porosity, it can be classified as a microfiltration or ultrafiltration
process. Microfiltration membranes have pore sizes typically between 0.1
µm and 10 µm. Ultrafiltration membranes have much smaller pore sizes and
are classified based on cutoff, which is based on their ability to retain the
protein with a known molecular weight (i.e., 1 KD to 1000 KD). A rule of
thumb is to select the three- to five-times smaller membrane cutoff than the
molecular weight of the protein to be retained. TFF is commonly used for
concentration, desalting, buffer exchange, and fractionation of proteins.
Until recent years there were limited applications of TFF in cell harvest and
cell lysate clarification. This case study describes the possible use of TFF as
an alternative to centrifugation for cell harvest and cell lysate process.
Centrifuges are commonly used for cell lysate clarification or for the
cell harvest purposes because of their ease of use at small scale and faster
processing time. However, the story changes quite dramatically when it
comes to scale-up because of the complex relationship between the lab-scale
or bench-top centrifuge to the large-scale industrial centrifuge. The same is
applicable when the centrifuge needs to be scaled down for validation or
process optimization purposes. On the other hand, TFF can be easily scaled
up or scaled down by keeping the channel length and channel height
constant. This becomes very important for current good manufacturing
practices (cGMP) contract manufacturing companies like QSV Biologics,
where technology transfer and process development is very important.
The disadvantages with TFF membranes are that they foul rapidly,
resulting in low flux and throughput for cell lysate processes. This can result
in a system with a huge membrane area and increased cleaning burdens,
especially when reusability is a requirement, making TFF a very costly unit
operation compared to centrifugation. So the question is, can we use TFF for
an application such as cell lysate clarification?
An argument for the possible usage of TFF for applications like cell
lysate clarification becomes stronger with the new generation of membranes.
These membranes, which have an ability to handle high back pressure, high
∆P, and can be operated at high transmembrane pressure (TMP), resolve
some of the issues and provide an efficient process with high throughput.
The advantage of using TFF at this initial stage is to simultaneously achieve
clarification and concentration very early in downstream processing (DSP).
By concentrating the protein of interest, TFF promotes the reduction of
pump size, buffer volumes, storage vessel sizes, etc. for subsequent
downstream unit operations. If the next unit operation is chromatography,
assuming the matrix has a high binding capacity, using TFF before the
column can reduce processing time, column size, and matrix volume, which
can result in significant cost savings. In addition, incorporating TFF will also
ensure an increase in column lifetime and column performance.
Another typical observation is that TFF requires higher volumes of
buffers for cell lysate washing compared to centrifugation, but this depends
on the rejection coefficient of the membrane for the product of interest and
the impurities. Rejection coefficient can be calculated by analyzing the feed,
retentate, and permeate streams. One solution to high buffer usage is a
membrane with low rejection for the protein of interest and high rejection
for the impurities, when impurities need to be retained. Another solution is
to use a cascade TFF unit operation. Cascade TFF unit operation can lower
buffer requirements because permeate of the following unit operation is a
feed to the former TFF unit, assuming that the protein is retained at the
second unit of cascade. The following case study will explain the
purification process of recombinant proteins produced in inclusion bodies
(IB).
CASE STUDY
A process flow diagram for IB purification is shown in Figure 1. Escherichia
coli cells containing IB were retained using a Millipore Pellicon 2, 0.22 µm
and collected for cell lysis. Harvested E. coli cells were then lysed using a
pilot-scale, high-pressure homogenizer. E. coli cell lysate containing IB was
loaded on a Millipore Biomax 1000 KD to remove the small proteins and
low-molecular weight impurities. IB dissolution was then performed using
Biomax 1000 KD under recirculation of Guanidine chloride for three hours.
The soluble protein was loaded on the same regenerated 1000KD membrane,
and the protein of interest was collected in permeate, leaving cell debris and
other impurities in retentate. Protein was recovered in the permeate stream at
the conclusion of the process, analyzed, and compared for purity with the
centrifugation process.
JOSH, PLACE FIGURE 1 HERE.
Bottino, Chaudhry, Sourrirajan, and others have published many
research papers and articles on the impact of membrane pore size,
recirculation rate, permeate pressure, and TMP on the performance of the
filtration process. All of these play a significant role in reducing the fouling
of the membrane. TFF can significantly affect process performance and
yield, i.e., process operations with high TMP, low recirculation rate, and no
permeate pressure will produce low yield and also foul rapidly. Processes
operating at room temperature have high flux compared to processes at low
temperature due to the change in viscosity with temperature. For high
throughput processes, one should maintain a minimum crossflow rate. The
membrane vendor normally provides the minimum recirculation rate.
In this case the recirculation rate was maintained at 30 L/m2/min,
based on the Millipore specifications for Pellicon 2. If the minimum
recirculation rate is not maintained, the membrane will start building a gel
layer, which will foul the membrane very quickly and potentially choke the
pores irreversibly. We observed that flux was reduced from 55 ml/min to 30
ml/min during one hour of processing time. To reduce fouling, the permeate
pressure was increased and adjusted to maintain constant TMP. The
selection of TMP was determined by applying different TMP and ∆P on the
membrane. The ideal TMP was selected based on the initial fouling
condition from the TMP vs flux graph shown in Figure 2. During the
process, TMP was kept almost constant to observe the fouling pattern. As
the procedure continued, flow rate further decreased to 20 ml/min. It is not
uncommon to use at least five diafiltration volumes of buffer if not more.
These buffer requirements for TFF can be higher than the centrifuge process
when operated as a stand-alone unit operation without cascade arrangement.
JOSH, PLACE FIGURE 2 HERE.
Figure 1. IB Purification Process from E. coli Cell Lysate
(Josh, Please use Figure 1 from separate file as that’s the one I
corrected. Please make whatever spacing, color changes, etc. you want.)
Homogenization
Microfiltration
0.22 um
E.Coli Cell harvest
Protein of interest
Ultrafiltration
1000 KD
IB harvest
&
Dissolution
(PLACE UNDER FIGURE 1) Increasing TMP increases the flux.
However increase in flux after a certain TMP is not significant and often
leads to rapid fouling. In this case a TMP of 4.5 psi is the ideal value to
choose for a high-throughput process.
Table 1. TMP (Pounds per Square Inch [PSI]) vs Flux (Liters per Square
Meter per Hour [LMH]) Data
(JOSH, AUTHOR PROVIDES TABLE 1 IN SEPARATE FILE IF THAT
HELPS YOU. (NO TITLE IN THAT FILE.)
TMP
PSI
0
3
4
4.5
5
6
in Flux
in
LMH
0
574.6
639.9
681.1
682.6
679.8
Figure 2. TMP vs Flux for Pellicon 2 for Cell Lysate Process
(JOSH, GRAPHIC IN SEPARATE FILE HAS BEEN FIXED. PLEASE
USE THAT ONE.)
Flux in LMH
Pre ssure Excursion Graph for P2M ini 0.22 u V Scre e n
Optimum TMP
800
700
600
500
400
300
200
100
0
0
1
2
3
4
5
6
7
TMP in PSI
(PLACE UNDER FIGURE 2)
Figure 2 shows the linear relationship between flux and TMP. Pressure
difference is a driving force for membrane processes; increased pressure
results in an increase in flux as per Darcy’s rule for filtration. The neck of
the curve is considered to be the ideal operating condition for TFF
processes.
Figure 3 shows the fouling characteristics of the membrane during cell
lysate and IB retention. The membrane fouls rapidly after 50 minutes of
processing. This fouling is due to high solid concentration in the retentate
stream, which increases further during the process. Fouling can be reduced
by starting the buffer wash early or by using a cascade TFF system. Similar
results were observed for the ultrafiltration (UF) step during IB dissolution
and protein recovery in permeate.
Figure 3. Flux and TMP vs Time for 1000 KB Biomax During Cell Lysate
and IB Retention
Flux of the Process at Given TMP
E. coli IB Harvest with P2Mini 1000KD, V Screen
50
4.5
45
40
4
Diafilrations Started
35
3.5
TMP in psi
Flux in LMH
30
25
20
3
15
10
2.5
5
0
2
0
6
10
13
16
22
27
30
36
49
57
62
94
98
111
136
TIME (min)
Another example of the E. coli cell lysate process is shown in Figure 4,
which compares a process using several precipitations and centrifuge step
with the TFF process. The TFF process appears simpler and more straight
forward.
Figure 4. Comparison of Initial Clarification Processes with Centrifuge (A)
and TFF (B) (JOSH, CORRECTED FIG 4 IN SEPARATE FILE)
Process (A)
Homogenizer
Feed
Centrifuge
Precipitation
Centrifuge
Precipitation
Centrifuge
MF
NFF
UF/DF
Chromatography
steps
Process (B)
Homogenizer Feed
MF
0.22 um
UF
300 KD
UF
30 KD
Chromatography
steps
Retentate stream
Process (A) consists of several centrifuge and precipitation steps that
are replaced by TFF in process (B). For proteins with a molecular weight
between 100 to 150 KD, 30 KD UF membrane may be used. It is ideal for
the cascade process, providing higher retention and high yield during
concentration and buffer exchange steps. The flow schematic of process (B)
can fractionate the protein of interest from impurities and can concentrate it
to the desired level before loading to the chromatography column for further
purification. The advantage of process (B) is reduced DSP with fewer steps
resulting in similar or better performance compared to process (A). The
fouling is reduced due to continuous dilution of feed by dilution buffer. The
majority of this dilution buffer comes from the cascade loop. It is important
to run all the systems at the same permeate flow rate. The use of a two-stage
cascade system is much simpler and easier to control due to fewer process
complications.
Conclusion
It was evident from the above examples that TFF can be used for cell lysate
processing. Effective process design may require a series of small
experiments to identify the optimum processing condition, which can result
in an economical process with high yield. It was concluded that further trials
will yield additional improvements for TFF as an alternative to centrifuge
for cell lysate clarification. Using permeate pressure and optimum TMP is
key to reducing membrane fouling. The cascade TFF system may be used to
reduce buffer requirements and to achieve efficient impurity clearance or
protein recovery.
ACKNOWLEDGMENT
I would like to express my sincere thanks to Dr. Graeme Macaloney and Dr.
Shoand Chaterjee for their assistance in the preparation of this case study.
Jignesh Padia, QSV Biologics Ltd.
References
1. Bioprocess Engineering, Chapter 4 “Tangential Flow Filtration Systems
for Clarification & Concentration” by Eric Rudolph & Jeff Macdonald,
John Wiley Publication.
2. Bioprocess Engineering Principles, Chapter 10 “Unit Operation” by
Pauline Doran, Academic Press
3. Millipore Application note AN1045EN00: Clarification of recombinant
protein using TFF as a cascade operation.
4. Millipore Technical Brief TB032: Protein Concentration and Diafiltration
using TFF.
5. Effect of Operating Conditions on the Performance of the Membrane. by
Bottino etal. J. of Mem. Sci.
6. Determination of Interaction Forces and Average Pore Size and Pore Size
Distribution and Their Effects of Fouling of Ultrafiltration Membranes.
By Sourirajan etal.
7. Membrane separations in biotechnology, Biochemical Engineering by
Robert Van etal.
8. Membrane separations: range of options by M. Estabrook and M.
Beauchemin Amersham Biosciences,
9. Influence of processing conditions on the properties of ultrafiltration
membranes, J. of Mem. Sci. by Chowdhury etal.
10. Troubleshooting tangential flow filtration, CEP Magazine by Kent
Inversion