3. Optimised cloning procedure for fast clone recovery

Just as host cell growth rates can be speeded up, a similar approach can be used to create faster growth rate during the cloning procedure and high cloning efficiency. Increasing the cloning efficiency in serum free medium from typically 30% to 60% will yield more clones for identification of the optimal producer or will reduce the number of plates that have to be screened. In addition, the time required to expand single cell derived clones is one of the currently unchangeable bottlenecks of cell line development. Our preliminary results show that cells produce and secrete factors, which provide a favourable matrix that stimulates other cells to grow. Likewise, as viability declines, such secreted or released factors contribute to the death of other cells. This decreases the survival of resistant cells during selection. Traditionally, especially with serum free media, a mixture of fresh medium and sterile filtered culture supernatant has been used to enhance clone growth in serum free medium. We postulate that such defined secreted factors of CHO cells can be used to stimulate clonal colony formation, which in combination with the high speed host cell line established in Project 1, will enhance the speed of cell line development.

Approaches:

  • Analysis of secretome of CHO cells
  • Controlled addition of identified factors and evaluation of effects on cloning efficiency and time


Target:

  • Defined media additives for enhanced clone growth and cloning efficiency
  • Increased cloning efficiency
  • Faster clone development for expansion