Austria VWR International GmbH Graumanngasse Wien Tel.: Fax:

Size: px

Start display at page:

Download "Austria VWR International GmbH Graumanngasse Wien Tel.: Fax:"

Margaret Jodie Gregory
6 years ago
Views:

Austria VWR International GmbH Graumanngasse 7 1150 Wien Tel.: 01 97 002 0 Fax: 01 97 002 600 E-mail: info@at.vwr.

1 Austria VWR International GmbH Graumanngasse Wien Tel.: Fax: info@at.vwr.com Belgium VWR International bvba Researchpark Haasrode 2020 Geldenaaksebaan Leuven Tel.: Fax: customerservice@be.vwr.com Denmark VWR - Bie & Berntsen Transformervej Herlev Tel.: Fax: info@dk.vwr.com Finland VWR International Oy Valimotie Helsinki Tel.: Fax: info@fi.vwr.com France VWR International S.A.S. Le Périgares Bâtiment B 201, rue Carnot Fontenay-sous-Bois cedex Tel.: (0,15 EUR TTC/min) Fax: (0,15 EUR TTC/min) info@fr.vwr.com Germany VWR International GmbH Hilpertstrasse 20a D Darmstadt Tel.: * Fax: * info@de.vwr.com *0,14 /Min. aus d. dt. Festnetz, Mobilfunk max. 0,42 /Min. Hungary VWR International Kft. Simon László u Debrecen Tel.: (52) Fax: (52) info@hu.vwr.com Ireland / Northern Ireland VWR International Ltd / VWR International (Northern Ireland) Ltd Orion Business Campus Northwest Business Park Ballycoolin Dublin 15 Tel.: Fax: sales@ie.vwr.com Italy VWR International s.r.l. Via Stephenson Milano (MI) Tel.: Fax: info@it.vwr.com The Netherlands VWR International B.V. Postbus AD Amsterdam Tel.: Fax: info@nl.vwr.com Norway VWR International AS Haavard Martinsens vei Oslo Tel.: Fax: info@no.vwr.com Poland Labart Sp. z o.o. A VWR International Company Limbowa Gdansk Tel.: Fax labart@labart.pl Portugal VWR International - Material de Laboratório, Lda Edifício Neopark Av. Tomás Ribeiro, 43-3 D Carnaxide Tel.: Fax: /9 info@pt.vwr.com Spain VWR International Eurolab S.L. C/ Tecnología 5-17 A-7 Llinars Park Llinars del Vallès Barcelona Tel.: Fax: info@es.vwr.com Sweden VWR International AB Fagerstagatan 18a Stockholm Tel.: Fax: info@se.vwr.com Switzerland VWR International AG Lerzenstrasse 16/ Dietikon Tel.: Fax: info@ch.vwr.com UK VWR International Ltd Customer Service Centre Hunter Boulevard Magna Park Lutterworth Leicestershire LE17 4XN Tel.: Fax: uksales@uk.vwr.com Go to vwr.com for the latest news, special offers and details of your local VWR distributor. EN VTDSVZ

Quantitative Western blotting utilising new horizontal gel system I

2 Issue 27 Autumn 2011 the market source for life science I Page 10 Real-time PCR analysis of surfactin gene expression I Page 25 Quantitative Western blotting utilising new horizontal gel system I Page 30 Batch and fed batch cultivation of different mammalian cell lines

the market source for life science Batch and fed batch cultivation of different mammalian cell lines in the BIOSTAT Aplus bioreactor D. Lampe, B. Pütz, D.

3 the market source for life science Batch and fed batch cultivation of different mammalian cell lines in the BIOSTAT Aplus bioreactor D. Lampe, B. Pütz, D. Lütkemeyer, Frank Gudermann, Apparative Biotechnology, Department of Engineering Sciences and Mathematics, Bielefeld University of Applied Sciences, Bielefeld, Germany Sabrina Armgart, Ute Noack, Sartorius Stedim Biotech GmbH, Goettingen, Germany BIOSTAT Aplus is a compact laboratory bioreactor with an autoclavable vessel featuring a stirred tank design. The system is available in a choice of culture 1, 2 and 5 litre volumes and preconfigured versions for microbial and cell cultivation. This wide selection makes it easy for users to choose the bioreactor model that best matches their specific needs. Thanks to the intuitive operating design of this bioreactor via notebook PC, it is especially suitable for educational purposes or first time bioreactor users. Typical applications range from small scale protein expression to up-scale experiments from uncontrolled shake or T-flasks to controlled cultivation conditions. All systems measure and automatically control dissolved oxygen (DO), ph, temperature, foam and level. In particular, DO control can be set-up as in larger scale bioreactors normally used in process development, while keeping operation easy. Stirrer speed, gas mixing and substrate addition can be selected for the DO cascade. Automatic ph control can be accomplished by selecting acid and base addition or CO 2 and base addition to suit the needs of a specific application. Three internal peristaltic pumps can be used for corrective agents or substrate. A fourth external pump can also be connected. Different impeller and sparger designs are available to suit organisms with a high demand for oxygen as well as sheer sensitive cells. The BIOSTAT Aplus also includes an installation video and cultivation recipes for common cell lines to facilitate installation and first time use of the bioreactor. The following application data was compiled at the University of Applied Sciences in Bielefeld, Germany. At Bielefeld University the BIOSTAT Aplus is used for various applications during students practical lab course work in cell cultivation. 1st application: CHO cultivation in fed batch mode For fed batch cultivations in a 2 litre BIOSTAT Aplus system, a CHO cell line was used which secretes a recombinant monoclonal IgG antibody. The protein-free, chemically defined medium, MAM-PF 2 from BioConcept Amimed, was used as the culture medium. The concentration of living and dead cells was measured using the Cedex from Roche Applied Sciences. The concentration of glucose, lactate, glutamine, ammonium and IgG was determined using the Cubian XC from Optocell. (Figure 1) The bioreactor was equipped with a 3 blade segment impeller with pull down mixing direction in the upper section and a 6 blade disc (Rushton) impeller approx. 2 cm above the sparger. At an agitation speed of 200 rpm, this configuration in combination with the microsparger used for the submerse gassing ensures a small gas bubble diameter as well as good mixing conditions. The residence time of the small gas bubbles in the medium is also 30 I VWR International I VWRbioMarke Issue 27 I September 2011

Cell biology For more information on these products contact your local VWR sales office, send an e-mail to vwrb

4 Cell biology For more information on these products contact your local VWR sales office, send an to or visit our website Figure 1: Schematic diagram of the BIOSTAT Aplus for fed batch cultivation increased, as they are retained for some time in the lower section of the bioreactor due to the flow profile of the disc impeller. The regulation of the ph at 7,1 using CO 2 was performed exclusively via the headspace. A constant flow of air (200 ccm) through the headspace was mixed with the corresponding proportion of CO 2 via the control of the Aplus. Using sodium carbonate (1 M), the ph could, if necessary, be maintained in the alkaline range during the second phase of the cultivation. The required amount of dissolved oxygen was transfered into the suspension by submerse gassing using the microsparger. Pure oxygen was used to minimise the gas flow through the liquid. In case of dissolved oxygen concentrations below the target value of 40%, a short injection of oxygen was used to generate a succession of bubbles, which were held in the liquid for some time. In this way, a continuous gas flow through the sparger was avoided and pulsed gassing of the culture was achieved. As a result foaming on the surface of the liquid was significantly reduced, minimising the need for anti-foam agents (Antifoam C, Sigma). (Figure 2) The Figure shows a fed batch cultivation with the addition of separate 25 ml doses of CHO Feed Bioreactor Supplement from SAFC Biosciences. The feeding was performed every 48 hours. Additionally the glutamine and glucose concentration of each sample were determined with the Cubian XC, Optocell. The substrate need for the next 48 hours was calculated based on the results. Furthermore, the specific growth rate µ as well as the specific uptake rate qglc and qgln were approximately determined from the concentration of the last two samples. The addition times of the substrate mix are marked in the graph by grey bars. With this strategy, a maximum viable cell density of cells/ml with a viability of 90% was achieved on the seventh day. In a second fed batch cultivation using the same cell line, the viable cell density was additionally determined online by continuous measurement of the capacity of the cell suspension using the i-biomass 465 from Fogale. In this second scenario, the aim was to keep the cell culture at a constant cell concentration for as long as possible, as the cell line only produces noteworthy amounts of antibodies in the stationary phase. In this process, 50 ml of amino acid concentrate and 50 ml of glucose/glutamine mix the composition was adapted to the requirements of the culture in advance were added at three separate times. The times of these additions are once again marked by grey bars in the following two graphs. (Figure 3) Figure 2: Development of cell concentration and viability over time Figure 3: Variation of cell and product concentration over time with feed times VWR International I VWRbioMarke Issue 27 I September 2011 I 31

the market source for life science The viable cell concentration reached 56 10 5 cells/ml with a viability of 98%. The specific growth rate was 1.06 d-1.

(Figure 4) In order to continuously determine the concentration of viable cells, the capacity of the cell suspension was measured using the i-biomass 465.

After the third addition of the concentrate and a slight reduction in the concentration of viable cells the results of the two measuring processes start to differ from one another.

5 the market source for life science The viable cell concentration reached cells/ml with a viability of 98%. The specific growth rate was 1.06 d-1. At the end of the process, the product concentration was 35 mg/l (referred to as 100% in Figure 3). (Figure 4) In order to continuously determine the concentration of viable cells, the capacity of the cell suspension was measured using the i-biomass 465. Figure 4 shows that, in the first 5 days of the cultivation, the capacity correlates very well with the concentration of living cells determined offline. After the third addition of the concentrate and a slight reduction in the concentration of viable cells the results of the two measuring processes start to differ from one another. (Figure 5) The substrate concentrations of the glucose and glutamine were kept between 2 g/l 6 g/l (glucose) and 1.5 mm 10 mm (glutamine) during the cultivation by the addition of concentrates. On the eighth day, the lactate concentration rose to 7 g/l. 2nd application: BHK cultivation in batch mode In a conventional batch process, the aim was to achieve a maximum viable cell concentration without additional substrate feeding. For this purpose, a BHK cell line was cultivated in a chemically defined medium, specially designed for this task by Teutocell. The culture volume was 1 litre. A 6 blade disc impeller was used for the mixing and a ring sparger was used for gassing. In the same way as in the cultivation described above, the agitation speed was set at 200 rpm and the regulation of the ph at the target value of 7.1 was achieved using CO 2 exclusively via the head space. The DO set point was set at 40% air saturation was achieved using pure oxygen by submerse gassing using the ring sparger. In this way, it was possible to achieve a concentration of viable cells of cells/ ml. However, on the third day, the glutamine concentration of the cultivation was 2.5 mm. The grey bar in Figure 6 shows that, at this point, the substrate concentration in the bioreactor was increased to over 6 mm by the addition of a glutamine concentrate. (Figure 6) Summary The results of the batch and fed-batch cultivation of animal cells show that the compact and easy-to-use BIOSTAT Aplus can be used very effectively in teaching. The system is not only outstandingly suitable for batch operation, in which cell concentrations of over 13 million cells per ml are achieved, it can also carry out fed-batch processes over a period of more than a week. Using a corresponding adapter (Sartorius Stedim Biotech), additional electrodes besides ph, DO, PT100 and antifoam can be installed for online measurements via the top plate of the bioreactor. In this example, the capacity of the cell suspension was measured online and compared with the cell concentrations determined offline. Figure 4: Correlation between the concentration of living cells and the capacity of the suspension Figure 5: Variation in the concentration of glucose, lactate and glutamine over time Figure 6: Variation in cell concentrations over time 32 I VWR International I VWRbioMarke Issue 27 I September 2011

All you need for cryo storage

All you need for cryo storage Performance, safety and reliability vwr.com Preserving your life s work Table of contents Canister storage systems Box storage systems Dry shippers Liquid nitrogen dewars