Investing for the Future: Quality cGMP Cell Banks

Cell banking is essential for a biological product manufactured by the fermentation process, such as plasmid DNA. Particular emphasis is given to cGMP cell banks by following the regulatory standards (21CFR610). But how do you generate high quality cGMP cell banks, and what are considered important attributes? Guidelines do not tell us.

First, let’s look at the two-tiered bacterial cell banking system:

  • Seed stock –> Master cell bank (MCB) –> Working cell bank (WCB)

Seed stock is established from a single colony (or clone), MCB is generated from the seed stock to ensure the genetic integrity within cells, and WCB is produced from MCB and used for production. A multitude of characterization assays are then performed, such as identity, purity, phage, cell viability, plasmid retention, copy number, sequencing, restriction analysis, etc.

Although the process seems straightforward, is it sufficient to simply pick a single colony and grow it up for use as an MCB and WCB? No. (Believe it or not, that is what most research and manufacturing organizations do).

It’s important to perform extensive screening on the seed stock prior to generating cGMP cell banks. Additional screening on the seed stock will directly impact the quality of the final cGMP product. Below is the VGXI cell banking system:

  • Seed stock –> Screening –> Master Cell Bank (MCB) –> Working Cell Bank (WCB)

Why extra screening? Two simple reasons: Quality and Quantity.

Quality

A plasmid may assume dynamic structural conformations: supercoiled (SC), open circular (OC), relaxed, linear, dimer, multimer, deletion, etc. The quality of a plasmid is generally acknowledged as the SC percentage, or more specifically, the SC monomer percentage. Therefore, rational and efficient design of the vector sequence is vital. Dimer and multimer forms for a plasmid of large size or with repetitive DNA sequences have been frequently observed. During the screening process, colonies of pure dimers or deletions are able to be eliminated. Even if all selected colonies assume correct size, colonies can be screened for high SC monomer percentage.

Quantity

Plasmid yield, or copy number of MCB/WCB, will affect the production process. Yet, based on experience, plasmid copy number of individual colonies is not solely controlled by the replication of origin, and not all host/vector systems have homogeneous copy numbers for all single colonies. So regarding the quantity of your cell banks, you may need to answer two questions:

(1) Did I pick the colony of the best yield (shaker flasks)?

(2) Will I still get the highest yield in fermentation (bioreactors)?

Over 4-fold yield differences among single colonies have been observed when tested in shaker flasks. Furthermore, yields in shaker flasks frequently do not correlate with high cell density fermentation in the bioreactors. For one plasmid tested, 2-3 fold differences in specific and volumetric yields were shown for two seed stocks which had similar yields in shaker flasks.

Overall, generating high quality cGMP cell banks requires a lot of work but is an investment for the future; it will demand less optimization of the fermentation process and improve product purity for downstream processing.

-Ying C.

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