The document summarizes optimization work done using FILTROX's FILTRODISCTM BIO SD filtration system to separate cell debris from recombinant E. coli fermentation broth. Key findings include: (1) Precoat filtration using diatomaceous earth filter aid Celpure® C65 achieved the lowest turbidity in filtrate; (2) Increasing precoat concentration and volume further reduced turbidity; (3) Precoat filtration allowed high protein passage while effectively retaining cell debris. Further work may optimize precoat formation and examine particle size distribution and endotoxin levels.

1. Cell Debris Removal by
Single-Use
Diatomaceous Earth Filtration
9th BILS, Berlin 10th & 11th February 2016

2. The FILTROX Group
The FILTROX Group is an international Group of Filtration Companies
headquartered in St. Gallen, Switzerland and 100% privately
owned
It started in 1938 with the foundation of FILTROX Filter-Werke AG in
the Sitter Valley in St. Gallen
First in the industry to introduce body feed technology in
single-use format
Focused on depth filtration technologies in Pharma, Biotech as well as
in Food & beverage industries, single-use and not single-use
Today, the FILTROX Group consists of 10 companies in 9 different
countries a total of approximately 250 employees

3. Objectives
1. Optimization of separation of cell debris
2. Protein content in filtrate
3. Outlook
FILTRODISCTM BIO SD
2" Capsule
FILTRODISCTM BIO SD
12" double module FILTRODISCTM BIO SD
Skid

4. Optimization of separation of cell debris
Biomass
Recombinant E.coli producing the enzyme Cytochrome P450
- Buffer: Phosphate buffer (pH 7.5)
- Equipment: Niri Soavi Panda 2K
- Pressure: ~1000 barg
- Duration: 15 min
- Efficiency of rupture: ≥99%
- Concentration of debris after rupture: ~25 g/L (wet biomass)

5. Optimization of separation of cell debris
magnetic stirrer balance
FILTRODISCTM BIO SD 2" Capsule
peristaltic pump: Ismatec MCP,
pump head Masterflex Easy-Load II
inner tube diameter: 6,4 mm
Test Setup

6. Optimization of separation of cell debris
Filter
Capsule
Filter
Aid
FILTRODISCTM
CH
09P
Celpure®
C
65
FILTRODISCTM
CH
73P
Celpure®
C
100
FILTRODISCTM
CH
103P
Celpure®
C
300
FILTRODISCTM
CH
143P
FILTRODISCTM
CH
153P
Celpure® = registered by Advanced Minerals
Used Material

7. Optimization of separation of cell debris
Used Material
FILTRODISCTM BIO SD is a depth filter based filter system
designed for cell harvest and cell debris removal.
It start with a 2” capsule and ends with a 16” double module.
Celpure® is a pharma grade diatomaceous earth filter aid.
Celpure® = registered by Advanced Minerals

8. Depth filtration Alluvium filtration
Optimization of separation of cell debris
Surface filtration
Depth Filtration Functional Principle

9. Clogging Curve
Depth Filtration vs. DE Filtration
Optimization of separation of cell debris

10. Optimization of separation of cell debris
Optimal flow rate:
Can be kept during the whole filtration time
Turbidity measurements:
- OD600 is used
- Measured absorbance is caused in this case by
cell debris
- Used to
- Characterize the filtrate samples
- Compare the different filter types and
filter aids
Flow rate / turbidity measurements

11. Optimization of separation of cell debris
Filtration
number
Filter sheet
type
Filter aid type OD600 [-]
2 CH 73P Celpure® C300 0.118
6 CH 73P Celpure® C100 0.075
4 CH 73P Celpure® C65 0.056
Conclusion:
Celpure® C65 gave the lowest turbidity content
Selection of filter aid

12. Optimization of separation of cell debris
Selection of filter sheet type
Filtration
number
Filter
sheet type
Retention
rate [µ]
OD600 [-]
10 CH 103P 1.5 – 0.6 0.183
16 CH 143P 0.4 – 0.2 0.111
17 CH 153P 0.2 – 0.04 0.107
For further trials filter sheet type CH 103P and
Celpure® C65 was chosen.
Questions:
- reduction of OD by
1. increasing concentration of filter aid?
2. use of precoat filtration
3. use of flocculants

13. Optimization of separation of cell debris
Variation of filter aid concentration
Filtration
number
Cfilter aid [g/l] OD600 [-]
8 25 0.0435
13 37.5 0.0445
Celpure® C65 and
CH 103P are used

14. Optimization of separation of cell debris
Precoat filtration
Precoat and
filtration are done
with Celpure® C65,
CH 103P is used as
filter sheet type
Filtration Cfilter aid [g/L] OD600 [-]
precoating filtration
8 - 25 0,044
12 12,5 25 0,013

15. Optimization of separation of cell debris
Precoat filtration
Conclusion:
- Significant reduction of OD600 in a short interval
Questions:
- To which values runs the OD600 at longer filtration time
- Is it possible to achieve further reduction of OD600 by using
an increased Celpure concentration while precoating?
(filtration 18)

16. Optimization of separation of cell debris
Precoat filtration
Celpure® C65 and
CH 103P are used
Filtration Vprecoating[mL] Cfilter aid [g/L] OD600
precoating filtration
8 - - 25 0,0435
11 200 25 25 0,013
18 400 25 25 0,004

17. Optimization of separation of cell debris
Precoat filtration
Conclusion:
reduction of OD600 in a short interval as a result of:
- “slower” precoating
- Increased Celpure concentration
Best result:
Combination of “slower” precoating and increased
Celpure concentration
OD600 = 0.004 ≠ 0
OD600 of sterile filtrate = 0.004

18. Optimization of separation of cell debris
Protein content in filtrate
For the separation it is important to:
- reduce the turbidity of the filtrate by retention of the
cell debris
- ensure the unhindered passing of the proteins
through the precoat layer
Comparison of body feed filtration and precoat
filtration (concerning the protein concentration)
Filtration Cfilter aid [g/L] Vprecoating[mL] Vfiltration[mL]
Precoating Filtration
25 - 25 - 400
26 12,5 25 400 400

19. Optimization of separation of cell debris
Protein content in filtrate
- the precoat layer could retard the cell debris a long time
- proteins can pass the filter cake directly

20. Optimization of separation of cell debris
Conclusion
It is possible to separate cell debris in one step by use of
FILTRODISCTM BIO SD
best result: high protein content
low OD600 filtration
by use of precoat

21. Optimization of separation of cell debris
Outlook
• further optimization of all parameters while precoat filtration:
o optimization of the formation of the precoat layer
o optimization of Celpure® amount (precoating/filtration)
o higher concentration of cells in unfiltrate
o Influence of using flocculants like pDADMAC
o examination of particle size distribution in unfiltrate and
filtrate
• determination of endotoxin levels
• determination of enzymatic activity

22. Optimization of separation of cell debris
Acknowledgments
• Sandra Schaumburg, MSc
• Prof. Percy Kampeis
University of Applied Science Trier
Umweltcampus Birkenfeld
Bioverfahrenstechnik

23. Quality from Switzerland
Thank you very much for
your attention!
Questions ?
You are welcome to visit us
on booth #10