Bacteriophage amplification results using the CellMaker

One of the main applications of the CellMaker bioreactor system is bacteriophage amplification. We have been working with numerous companies and below we have detailed some examples of our successful work with phages.

 

Example 1

Cellexus carried out a demonstration at a clinical-stage company providing cost-effective cures for bacterial infections. As pH and dissolved oxygen monitoring were not points of interest, the 8L Regular CellMaker was used to look at amplification of the company’s proprietary phages.

Four litres of sterile TSB broth was pumped into the bioreactor, and 20 mL E. coli was inoculated after temperature was achieved.

 

Temperature: 37°C
Airflow: 5L/min

 

Time (mins)OD
00.010
300.025
600.084
750.153
850.227
1050.568

(Phage infection in green)

 

Phages were introduced after 85 minutes (in green), although the growth phase continued up to 105 minutes. After this point, the OD sharply declined and the solution went clear. The product was obtained by ultra-centrifugation and filtering through a 0.2 micron filter, and the final phage concentration was 2 x 1010 PFU per mL.

These are promising initial results for the first trial and are likely to be further improved with the addition of antifoam and optimisation to yield even better titres.

The CellMaker offers a fast and low-cost way to effectively manufacture phages for this biotech company.

 

Example 2

Cellexus provided a demonstration at a biotech company that develops CRISPR engineered antibacterial products to see how well the CellMaker could amplify their proprietary phages. 3.75L of sterile LB media was sterilised and pumped into an 8L Regular CellMaker with antifoam. E. coli was then inoculated and grown for 120 minutes.

 

Temperature: 37°C
Airflow: 5L/min
Time (mins)OD
50.01
300.01
600.05
900.19
1200.60

Phages were introduced at 120 minutes (in green) and the run was sustained for 4 hours.

The final concentration was recorded at 3.0 x 1010 PFU/mL. This was the highest amplification that the company has ever achieved with this phage and can be even further improved with process development.

In conclusion, the route to market in this instance is substantially reduced due to rapid E. coli growth and concentrated phage production.

 

Example 3

A North American biotech company ran two different fermentations on the CellMaker 8L Regular to look at amplification of their proprietary phages. Pseudomonas Aeruginosa was inoculated in the first run, and Staphylococcus aureus was inoculated in the second run. For both experiments, 4L of sterilised TSB broth was used and antifoam was added.

 

Temperature: 37°C
Airflow: 5L/min

Run 1: Pseudomonas Aeruginosa

Time (mins)OD
00.050
300.080
600.140
750.220
1050.400
1350.500
1650.400
1950.370
2250.250
2550.100

Run 2: Staphylococcus aureus

Time (mins)OD
00.001
900.070
1200.160
1350.230
1650.480
1950.710
2251.27
2850.53
3150.12

(Phage infection in green)

 

The first run gave a final PFU of 1.5 x 1010 per mL. The run also gave the same kinetics as a 100L batch already in production at the company—exemplifying the scalability of the CellMaker.

The second run was re-infected with phages after 225 minutes as the first infection was introduced late and at a higher OD. After clarification, the final concentration of the second run was 2.5 x 107 PFU/mL and also gave the same kinetics as a 150L batch already in production at the company.

In conclusion, the initial results were encouraging and can certainly be improved upon with relevant optimisation. The parallels in kinetics to larger production batches display the capability of scalable processes with the CellMaker.

 

Find out more about the CellMaker here, or find out more about bacteriophage amplification here.