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

Area of Science:

• Biotechnology
• Molecular Biology
• Bioprocess Engineering

Background:

• Single-stranded DNA (ssDNA) is crucial for genetic research and therapies.
• Mass production of ssDNA involves Escherichia coli secreting M13 bacteriophage-derived particles.
• Partial E. coli lysis complicates ssDNA purification using traditional methods like disc-stack centrifugation.

Purpose of the Study:

• To develop a scalable and efficient purification process for ssDNA phagemid particles.
• To overcome challenges in cell separation and particle recovery during ssDNA production.
• To replace traditional precipitation methods with advanced filtration techniques for pharmaceutical applications.

Main Methods:

• Tangential flow microfiltration for initial cell removal.
• Endonuclease treatment to enhance microfiltration efficiency.
• Cross-flow ultrafiltration with a 750 kDa cut-off for particle concentration and washing.
• Diafiltration for effective particle purification.

Main Results:

• Complete cell removal was achieved using tangential flow microfiltration.
• Endonuclease digestion improved phagemid particle transmission by over 90% without ssDNA degradation.
• Ultrafiltration successfully concentrated M13-derived particles by 10-fold while retaining them.
• The new process eliminates the need for polyethylene glycol 8000 and NaCl precipitation.

Conclusions:

• A scalable and efficient purification strategy for ssDNA phagemid particles has been established.
• This method enhances the suitability of ssDNA production for pharmaceutical manufacturing.
• The developed process offers a significant improvement over existing purification techniques.