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2.2 Cell Culture Methods

The sterilisation o f glass- and plasticware and inoculation vessels was performed in a Rodwell autoclave model Ensign (Hornchurch, U.K.) for 30 minutes at 120°C. Vessels below 2 1 and some plasticware were also sterilised in a Series 2100 media autoclave (Prestige Medical, Blackburn, U.K.). Batch suspension cultures were performed in 25, 125 and 250 ml Falcon shaker flasks (Becton Dickinson Labware, Plymouth, U.K.); 0.5 and 1 1 spinner vessels by Techne (Cambridge, U.K.) and 0.5, 1, 2.5 and 5 1 spinner vessels by Bellco (V.A. Howe & Co., Oxon, U.K.). All microcarrier cultures were performed in 0.5 1 Bellco vessels which were precoated with Sigmacote (Sigma) to prevent attachment of microcarriers to internal surfaces. All microcarriers were supplied by Pharmacia Biotech (St. Albans, U.K.) and prepared according to manufacturer’s instructions. The Cytodex 3 and Cytopore spinner cultures were agitated at 40 rpm and the Cytoline 1 cultures were agitated at 60 rpm. The individual properties of the microcarriers used are listed in Table 2-2.

Chapter 2.2 Cell Culture Methods

Table 2-2 Physical characteristics o f three different microcarriers.

M icrocarrier M atrix Size Density Pore Surface

(mm) _{(g m l ' )} size area

(pm) _g"*)

Cytodex 3 Dextran/ 0.18 1.04* N/A 0.46

collagen

Cytopore Cellulose 0.20-0.28 1.03* 30 1.1

Cytoline 1 Polyethylene/ length 17-25, 1.32 1-400 ?

silica thickness 4-11

* in 0.9% NaCl, N/A = not applicable because microporous, ? = unknown.

The fermentation system included a 15 1 LH (Inceltech U.K. Ltd., Reading, U.K.) glass-jacketed, single impeller bioreactor with a 2:1 aspect ratio. It has setpoint control of temperature by an LH controller and d02 and pH by an Anglicon controller (Brighton Systems, Brighton, U.K.). The temperature was maintained at 37°C (± 0.2°C), dO] was maintained at 40% o f air saturation (± 1%) and pH at 7.2 (± 0.05). Oxygen and pH probes were supplied by Mettler- Toledo (Greifensee, Switzerland) and the temperature probe by Brighton Systems. Oxygen was supplied by gassing the medium with air below the impeller with a 1 pm sintered glass sparger. Alkali addition to control pH was made using a 0.1 M NaOH solution delivered onto the surface o f the medium. Data-logging was supplied by Biotechnology Computer Systems, South Bank Technopark, U.K. run on a 386 DX personal computer (Viglen Ltd., Alperton, U.K.). The bioreactor was autoclaved in a BMM Weston/Drayton Castle ‘S’

Chapter 2.2 Cell Culture Methods

Series autoclave for 40 minutes at 120°C. The bioreactor system was used for batch experiments with daily sample volumes o f G.5-2.0 1 resulting in a 50% reduction of the working volume from 12 to 6 1. Larger samples were taken early in the experiment when the product titre was lowest; the agitation speed was stepped down in parallel with the working volume so that a constant power-to-volume ratio of 0.02 W 1'^ was maintained.

The fermentation system for fluidised bed culture was a 2 1 bioreactor (Cytopilot-Mini) obtained from Pharmacia Biotech. It was operated with setpoint control o f temperature and dOz and pH by two independent Anglicon controllers (Brighton Systems). The temperature was maintained at 37°C using an external Techne water bath (± 0.1 °C), dOi was maintained at 40% o f air saturation (± 2%) and pH at 7.2 (± 0.1). Ingold oxygen and pH probes were supplied by Mettler-Toledo AG. Data-logging was supplied by Biotechnology Computer Systems run on a 386 DX personal computer (Viglen Ltd.).. The bioreactor was autoclaved in a Rodwell autoclave model Ensign for 40 minutes at 118°C. The methods employed with the fluidised bed reactor as well as a schematic o f the bioreactor are covered in more detail in Section 3.7.

2.2.1 Static Gassing-Out Method

The theory behind this method is outlined in Section 1.3.4. Nitrogen gas was used to purge air from the medium by sparging beneath the moving

Chapter 2.2 Cell Culture Methods

impeller. When an anoxic condition was achieved air was sparged into the medium at a constant rate of 300 ml m in '\ The increase in dissolved oxygen concentration (Cl) was recorded every 3 minutes for 15 minutes or until the dissolved oxygen concentration had reached 40% of saturation. The temperature of the medium was maintained at 37°C.

2.2.2 Cell inoculum

All cell inoculums for 15 1 fermentation cultures were prepared in two identical 5 1 Bellco spinner vessels (Bellco). In each spinner vessel 2.5 1 of cells were cultured for 3 days or until the viable cell density had reached a mid-exponential density o f approximately 5 x 1 0 ^ cells m l'\ Two approaches were then employed to prepare the final inoculum. In most cases, the cells were allowed to settle for at least 30 minutes at 37°C and the top 2.0 1 of cell culture removed. The remaining 0.5 1 o f ‘concentrated’ cell culture (containing at least 60% o f the original cell number) was then transferred to a 2 1 inoculum vessel containing 1 1 o f prewarmed medium. Alternatively, after cell settling had been completed, the cell slurry was centrifuged at 1000 rpm for 5 minutes in 200 ml aliquots. The cell pellets were resuspended in prewarmed medium and transferred to a 2 1 inoculum vessel. For small scale microcarrier cultures cells were also seeded from cultures in the mid­ exponential phase o f growth. The total cell inoculum was added to half the final volume o f medium which had been preincubated with the microcarriers overnight. For a period o f 4 h the microcarrier cultures were agitated

Chapter 2.2 Cell Culture Methods

intermittently: 45 min on, 15 min off for each one hour period. After the 4 h the rest o f the medium was added and the spinner vessels agitated continuously at their respective agitation rates. Cell inoculation of the Cytopilot-Mini is detailed in Section 3.7 but was based on the second approach given above.

2.2.3 Cell banks

All working cell stocks were created from a master cell bank maintained at the University o f Kent. Cultures were tested every 8 months for the presence o f mycoplasma. Cells were grown for a minimum o f 6 passages because during early passages the viability o f the cell line was too poor for experimental use. The cells were also not cultured for longer than 20 passages because the CHO 320 cell line did not maintain a constant expression level (personal communication, P. Hayter).