Scalable Separation and Purification of M13-Bacteriophage-Derived Particles Containing Artificial Single-Stranded DNA...

Single-stranded DNA (ssDNA) solutions are promising innovations shaping the future of genetic research and medical therapies. In principle, biotechnological mass production of ssDNA can be achieved using Escherichia coli in a high-cell density fed-batch process by secreting phagemid particles derived from the filamentous M13 bacteriophages. However, the production of user-defined ssDNA enveloped in phagemid particles results in partial lysis of E. coli cells, affecting their sedimentation behavior, thereby complicating cell separation in disc-stack centrifuge processing. We have demonstrated that complete cell removal can be achieved using tangential flow microfiltration, although the M13 bacteriophage-derived particles, due to their 900 nm length and 6 nm diameter, pose a challenge for tangential flow filtration. Endonuclease-mediated nonspecific DNA digestion improved cross-flow microfiltration, enabling phagemid particle transmission above 90% without degrading the ssDNA product, which remained protected within the phagemid particle's protein shell. In a second cross-flow ultrafiltration, the M13-derived particles were concentrated by a factor of 10 and washed by diafiltration. A 750 kDa cut-off was found to be appropriate for ultrafiltration, quantitatively retaining the phagemid particles during concentration and diafiltration. With this step, the previously used addition of polyethylene glycol 8000 and NaCl for phagemid particle agglomeration and precipitation can be completely replaced, thereby making this new scalable purification process more suitable for pharmaceutical production.