Filtrox BILS 2016

Cell Debris Removal by Single-Use

Diatomaceous Earth Filtration

9th BILS, Berlin 10th & 11th February 2016

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

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

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)


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


Filter Capsule Filter Aid

FILTRODISCTM CH 09P Celpure® C 65



FILTRODISCTM CH 143P

FILTRODISCTM CH 153P

Celpure® = registered by Advanced Minerals

Used Material


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

Depth filtration Alluvium filtration


Surface filtration

Depth Filtration Functional Principle

Clogging Curve Depth Filtration vs. DE Filtration



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


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


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


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


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


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)


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


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


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


Protein content in filtrate

-  the precoat layer could retard the cell debris a long time

-  proteins can pass the filter cake directly


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


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


Acknowledgments

• Sandra Schaumburg, MSc • Prof. Percy Kampeis

University of Applied Science Trier Umweltcampus Birkenfeld Bioverfahrenstechnik

Quality from Switzerland

Thank you very much for your attention!

Questions ?

You are welcome to visit us on booth #10

Presentations & Public Speaking

Filtrox BILS 2016