Higher Diploma: Tangential Flow Filtration Practical Report

This practical report details an experiment on Tangential Flow Filtration (TFF), a membrane-based technique used for clarifying, concentrating, and purifying proteins. The report includes an introduction to TFF, comparing it with normal filtration and highlighting its applications in various fields like biopharmaceuticals. The methodology section outlines the materials and methods used, including the preparation of protein standards and the Biuret assay for protein determination. Results are presented in tables and graphs, showing extinction values at 540nm for different protein concentrations and calculating protein recovery percentages for retentate and permeate solutions. The discussion analyzes the results, comparing them to theoretical values and discussing factors affecting protein loss. The report concludes with a summary of the experiment, emphasizing the setup, process, and analysis of TFF, while also acknowledging the sources of error and success of the experiment.

Filtration using Tangential Flow
Practical on Tangential Flow Filtration Technique
Name of the Student :
Student Number :
Name of Lab partner :
Date of Practical :
1. Objective
Tangential Flow Filtration (TFF) techniques are membrane based and are used in
different applications like clarifying, concentrating, and purifying proteins.
2. Introduction
2.1 Tangential Flow Filtration (TFF)
In TFF the solution to be filtered is fed tangent to the filter’s surface where a difference
of pressure. As a result elements which are smaller in size in comparison to the pore-
size pass through the filter. Elements which are bigger than the pore size are retained and
flow back into the source reservoir (Figure 2.1). The concept of TFF is simple but its
correct application and execution requires in depth knowledge (Caraher, 2019).
Figure 2.1 Picture showing how TFF works (Pall.com, 2019)
2.2 Differences between TFF and normal direct flow filtration
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Filtration using Tangential Flow
The difference between TFF and normal filtration (commonly called direct flow or dead-
end filtration) as below.
 TFF filters is only membrane based, whereas normal flow filters can use
membrane, Paper or glass fiber to separate components during the filtration
process.
 In TFF, it is possible to recirculate the retentate whereas in. case of normal filter,
the process takes place usually in a single pass..
 In TFF, the retentate is a solution which can be used directly.. recovery of
retentate is virtually not available in normal flow filtration process and it requires
suspension to be collected, solution is to be made and processed again.
2.3 TFF application areas.
TFF finds its application in various fields like R & D, and production in the medical and
biopharmaceutical industries.
TFF are used in the following application areas:
o Desalt and concentrate pastes and proteins.
o Desalt and Concentrate Nucleic Acids [DNA / RNA / Oligonucleotides] (Casey et
al., 2011)
o Restoring and purifying antibodies from the media of cell culture.
o Purifying plasmid DNA (Rathore and Shirke, 2011) or chromosomal DNA from
blood sample. (Elmer, Harris and Palmer, 2011).
o Clarifying tissue homogenates or cell lysates.
o Removal of endotoxin from buffers, water and media solutions.
o Preparing the samples before column chromatography.
o Harvesting cells (Pattasseril et al., 2013)
o Recover or remove viruses.
2.4 What are the advantages of TFF
Advantages of TFF is that we can reuse the filter modules and the cost is also
comparatively low. Tangential Flow Filtration is used in many fields in biotechnology
particularly microbiology.
2.5 Process Flow Diagram of TFF
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Filtration using Tangential Flow
Conceptual diagram of a TFF System with Pump, Pressure Gauge, Retentate Screw
Clamp,
Reservoirs and Tubing Connections in shown in Figure 2.2 (Laboratory.pall.com, 2019).
3. Materials and Methods
Referred lab manual Section 5.3 page number 19 and 20. (Caraher,2019)
For preparation of protein Standard Curve Refer to Page no. 19 in Lab Manual. (Caraher,
2019)
For our practical the following reagents are mentioned which will be used for calculation
purpose
For Filtration of BSA solutions
1. The TFF unit is made ready in advance of the laboratory session.
2. 1000 ml of 1mg/ml BSA will be filtered. 10 ml is kept for initial protein analysis.
3. 750 ml of filtrate/permeate is collected – kept for protein analysis
4. When 250 ml of filtrate collected, the retenate is collected to analysis for protein
content
5. Protein standard (BSA 10mg/ml)
6. Biuret reagent (25ml per pair). Used as coloring reagent.
3.1 Table 1 shows the protein standard solutions in different dilutions .
Table 3.1 Showing four types of protein standard solution
Tube No. Protein Standard
sol. ( in ml)
Water ( in ml)
(distilled)
conc. Of protein
(mg/ml)
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Filtration using Tangential Flow
1 0 2 0
2 0.5 1.50 2.50
3 1.0 1 5
4 2.0 0 10
Protein concentrations are arrived at for tube 1 to tube 4 by the following method:
Protein concentration (mg/ml) is arrived at as follows:
Protein standard = 10 mg/ml
In case of tube 1 we have not taken any protein solution so protein conc. = 0 mg/ml
In case of tube 2 we have taken 0.5 ml of protein solution which contains =10*0.5=5 mg
of protein. Total volume of solution 2 ml. So protein conc.= 5/2=2.5 mg/ml.
In case of tube 3 we have taken 1 ml of protein solution which contains 10 mg of protein.
Total volume of solution = 1 ml + 1 ml = 2 ml. So protein conc. = 10/2 = 5 mg/ml
In case of tube 4 we have taken 2 ml of protein solution which contains 20 mg of
protein. Total volume of solution = 2 ml. So protein conc. = 20/2 = 10 ml/ml
3.2 Biuret Assay as Protein Determination Assay
3 ml of Biuret reagent is mixed in each tube.
The tubes are thoroughly mixed after the addition of the Biuret reagent and then the
tubes are placed in a 37oC water bath for 10 minutes. The tubes are removed from the
water bath and the extinction for each was read at 540nm using Spectrophotometer.
4.0 Results
4.1 The results are shown in Table 4.01.
Table 4.01 Showing Extinction at 540nm for different protein concentrations
TUBE Extinction using
spectrophotometer at 540nm
1 0.000
2 0.1888
3 0.388
4 0.744
BSA 0.085
Retentate 0.064
Filtrate 0.015
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Filtration using Tangential Flow
Table 4.02 showing protein concentration (mg/ml) against extinction @ 540 nm for
tube 1 to tube 4
Protein concentration mg/ml Extinction @ 540 nm
0 0.000 Abs
2.5 0.1888
5 0.388
10 0.744
Figure 4.01 Graph of Protein Concentration vs Extinction
0 2 4 6 8 1 0 1 2
0.00000
0.10000
0.20000
0.30000
0.40000
0.50000
0.60000
0.70000
0.80000
Extinction @ 540 nm
Protein concentration mg/ml
EXTINCTION
Using Table 4.02 data , a graph figure 4.01 was drawn with protein concentration
(mg/ml) as X-axis and Extinction as Y axis. The graph is passing through origin and is of
the nature Y=mX where m is the gradient and is expressed by Y/X= 0.744/10 = 0.0744.
We have to calculate the recovery of the protein after the filtration process.
1. For BSA solution : Sample contained 1mg/ml of protein,
Protein Concentration = Extinction / 0.0744 = 0.085/0.0744 = 1.1424 mg/ml
Original concentration = 1 mg/ml
So there is a little variation with standard 1mg BSA solution.
2. For filtrate Protein concentration = Extinction / 0.0744 = 0.015 / 0.0744 = 0.2 mg/ml
3. For retentate protein concentration = Extinction / 0.0744 =0.064 /0.0744 = 0.86 mg/ml
Percentage of recovery is calculated by the following method.
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Filtration using Tangential Flow
Dilution is a measure of Total volume divided by final volume
Theoretical yield is calculated by multiplying Dilution factor with Starting Material
Theoretical yield is shown in table 4.03
Table 4.03 showing calculation of theoretical yield (mg/ml)
Sample (ml) Dilution Factor Starting Material
( mg/ml )
Theoretical yield
(mg / ml)
1000 1 1.1424 1.1424
Permeate 750 1.33 1.1424 1.52
Retentate 250 4 1.1424 4.57
% recovery = (actual yield /Theoretical yield) × 100 % shown in table 4.04
Table 4.04 showing percentage of recovery for permeate and retentate solution. The BSA
recovery percentage is taken as 100 % as there is no filtration.
Actual yield Theoretical
yield
% recovery
BSA 1.1424 1.1424 100
Permeate 0.86 1.52 57
Retentate 0.2 4.57 4.4
5.0 Discussions
Retention percent of protein at retentate solution is found at around 57 % and the filtrate
solution at 4.4 %. The total is about 61.4 %. The loss of protein comes to 38 %. The
percentage of retention is low compared to the membrane standard of 90 %. However in
laboratory conditions the percentage of retention of 61.4 % is around 80% of the
theoretical efficiency which is a good value.
The molecular weight limit (MWCO) of a membrane is defined by its ability to retain a
certain percentage of a molecule in solution (typically 90% retention). In order to retain a
product, a membrane must be selected with an MWCO 3 to 6 times lower than the
molecular weight of the target protein (Laboratory.pall.com, 2019).
The loss of protein is because of various other factors.
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Filtration using Tangential Flow
A TFF process is controlled by a number of operating parameters which influence the
entire process (Karst et al., 2016). The most important parameters are
 Pressure at different points - fluid pressure and flow rates are important
parameters which control a TFF process.
 Flow rate at different points - Flow rates can be monitored at various points.
 Process Time - Process time.
 Filter Pore Size: The filter pore size regulates the process and if there is a
mismatch the filter becomes contaminated. Contaminations affect the effectivity
of the filter and reduce the length of time a filter can be used before formal
cleaning and servicing is required limiting the maximum processing capacity. The
choice of size of filter pore is important to minimize filter contamination. Smaller
pore filters are generally less prone to contamination as the particulate material
cannot penetrate and clog the pores.
 Area of the filter membrane. Volume of the filtrate generated depend on the
area of the membrane filter and the relation is expressed by
Membrane area (m2) = (Volume of filtrate generated in liter)/(Filtrate Flux Rate
(liter/m2/H)
 Protein binding: The extent of protein binding depends on the material used in
the filter and properties of the protein and increases when hydrophobicity
increases.. Protein binding though insignificant in laboratory experiments, can
cause a reduction in yield particularly for membranes with low NMWC
ultrafiltration, this may show error for filter contamination ( Callahan, Stanle and
Li , 2014). For diafiltration protein binding to the filter becomes a challenge when
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Filtration using Tangential Flow
trying to process low level protein. In such cases, it is better to choose a filter with
low binding tendency.
TFF technique is being used in downstream processing as the retention level of
protein through this method is satisfactory. This technique is used in downstream
processes for separation of components based on molecular size (Peters et al.,
2011) , concentration of product by removing solvent and small molecules before
further processing (Nicholson and Storm, 2011) and diafiltration.(Jungbauer,
2013). The TFF technique is also used for downstream processing for therapeutic
antibody production (Dizon-Maspat et al., 2011). The performance of this process
can be improved further by changing other factors in designing TFF process
which is listed below :
 Product yield
 Product concentration
 Selectivity – the importance of the level of purity of the product.
 filter stability and durability
 Downstream processing
6.0 Conclusion
Tangential flow filtration is an easy, efficient and fast method to separate and purify
biomolecules. In the experiment, the learning points are
a. To set up TFF filtration process in the laboratory.
b. To prepare the solution with different protein concentrations as standard solutions.
c. To run the filtration process with the sample protein solution by creating proper
experimental environment like pressure across the filter and time.
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Filtration using Tangential Flow
d. Collect the solutions from the permeate and retentate and perform the process on
Protein determination Assay using Biuret Assay. Observing extinction using
spectrophotometer @ 540 nm. To draw the curve using protein concentration vs
extinction for standard protein solutions.
e. To determine protein concentration for the unfiltered protein solution, permeate and
retentate and determine the percent of protein recovery from the permeate and
retentate solutions. Theoretically the sum of these two percentages should be 100 but
result has come about 62 %. The net loss i.e. 38 % is owing to different factors linked
to pressure across the membrane, time etc. and observation errors. But the
experiment is by and large successful.
References :
Callahan, D.J., Stanley, B. and Li, Y., 2014. Control of protein particle formation during
ultrafiltration/diafiltration through interfacial protection. Journal of pharmaceutical
sciences, 103(3), pp.862-869.
Caraher, , E. (2019). Higher Diploma in Pharmaceutical Manufacturing
Biopharmaceutical Processing PRACTICAL MANUAL 2019 Dr. Emma. Dublin: Dublin
University, pp.17-20.
Casey, C., Gallos, T., Alekseev, Y., Ayturk, E. and Pearl, S., 2011. Protein concentration
with single-pass tangential flow filtration (SPTFF). Journal of membrane science, 384(1-
2), pp.82-88.
Dizon-Maspat, J., Bourret, J., D'Agostini, A. and Li, F. (2011). Single pass tangential
flow filtration to debottleneck downstream processing for therapeutic antibody
production. Biotechnology and Bioengineering, 109(4), pp.962-970.
Elmer, J., Harris, D. and Palmer, A.F., 2011. Purification of hemoglobin from red blood
cells using tangential flow filtration and immobilized metal ion affinity
chromatography. Journal of Chromatography B, 879(2), pp.131-138.
Jungbauer, A., 2013. Continuous downstream processing of biopharmaceuticals. Trends
in biotechnology, 31(8), pp.479-492.
Karst, D.J., Serra, E., Villiger, T.K., Soos, M. and Morbidelli, M., 2016. Characterization
and comparison of ATF and TFF in stirred bioreactors for continuous mammalian cell
culture processes. Biochemical engineering journal, 110, pp.17-26.
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Filtration using Tangential Flow
Laboratory.pall.com. (2019). Introduction to Tangential Flow Filtration for Laboratory
and Process Development Applications. [online] Available at:
https://laboratory.pall.com/content/dam/pall/laboratory/literature-library/non-gated/id-
34212.pdf [Accessed 23 Mar. 2019].
Nicholson, P. and Storm, E., 2011. Single-use tangential flow filtration in
bioprocessing. BioProcess International, 9, pp.38-47.
Pall.com. (2019). Crossflow Technology. [online] Available at:
http://www.pall.com/main/graphic-arts/how-crossflow-filtration-works.page [Accessed
18 Mar. 2019].
Pattasseril, J., Varadaraju, H., Lock, L. and Rowley, J.A., 2013. Downstream technology
landscape for large-scale therapeutic cell processing. Bioprocess Int, 11(3), pp.38-47.
Peters, R., ten Dam, G., Bouwmeester, H., Helsper, H., Allmaier, G., vd Kammer, F.,
Ramsch, R., Solans, C., Tomaniová, M., Hajslova, J. and Weigel, S., 2011. Identification
and characterization of organic nanoparticles in food. TrAC Trends in Analytical
Chemistry, 30(1), pp.100-112.
Rathore, A.S. and Shirke, A., 2011. Recent developments in membrane-based separations
in biotechnology processes. Preparative Biochemistry and Biotechnology, 41(4), pp.398-
421.
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