Industrial Manufacturing of Plasmid DNA 학부 4 금현우 학부 3 강유리

Fig.1. cGMP manufacturing of pDNA flow chart Introduction  For manufacturing plasmid DNA, good manufacturing practice begins with prudent vector design

 cGMP (Current Good Manufacturing Practice)  Efficacy ( 유효성 )  Safety( 안전성 )  Stability( 안정성 )

ColE1 Plasmid  The choice of the plasmid’s origin of replication (ori) is critical for the plasmid copy number and hence for the cultivation titer.  Currently, the ColE1-derived, high copy-number pUC ori is widely established due to its reliably high copy number.  The introduction of random or defined mutations into the pUC ori can further increase the plasmid yield.

- -  Monocistronic Plasmids coding for one gene typically have about 5 kbp  Polycistronic coding for multiple genes may equal 8 kbp or larger

 The system utilizes the ori-encoded, copy-number controlling RNA I to silence a host-encoded repressor. This inhibition of the repressor (by antisense hybridization) allows the express- ion of an essential gene, which enables growth of the plasmid- carrying cells.

Selecting the Right Host Strain

 Yield and quality of pDNA are significantly influenced by the E. coli production host. Careful selection of an efficient host strain is therefore a critical factor right at the beginning of a pDNA development project.  Mostly, strains derived from E. coli K12, in particular DH1 or DH5- alpha, have been used for pDNA manufacturing. Some strains do not always fulfill the expectations regarding quantity (titer) and quality (supercoiled ratio).  As an alternative strain, E. coli K12 JM108 has been selected and successfully applied for high cell-density cultivation (HCDC). It has been demonstrated that E. coli JM108 consistently shows superior performance in small- and large-scale cGMP manufacturing.

Maximizing Titer By Upstream Optimization

 Another important aspect of fermentation is control of the specific growth rate µ. Fed-batch cultivation employing an exponential feeding regime is the most efficient mode for controlling µ in order to support enrichment of plasmid DNA in the biomass. Strict feed-rate control on a time basis, without plant operator input or a feedback-control mechanism, is the principal benefit of exponential and predefined nutrient addition.

Scale-Up Alkaline Lysis

HCDC (High Cell Density Cutivation)  Alkaline lysis is the first step in downstream processing of pDNA, followed by a series of chromatography and filtration steps.  The purpose of alkaline lysis is the release of pDNA from the biomass. 1 Lysis tube reactor 2 Neutralization loop reactor 3 Clarification device

Chromatographic Principle

Chromatography  From Greek χρώμα : chroma color γραφειν : graphein to write  Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures.  It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases.

HIC (Hydrophobic Interaction Chromatography)  Purification processes for plasmid DNA usually consist of up to three different chromato-graphic steps, each with specific features and purposes.  Hydrophobic interaction chroma-tography (HIC) is suitable for the first (capture) step. Particular focus has to be put on the optimization of the binding and elution conditions.

AEC (Anion Exchange Chromatography)  Ion exchange chromatography uses ion exchange mechanism to separate analytes.  Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained.

SEC (Size Exclusion Chromatography)  Size exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size.  Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster. Smaller molecules<<Larger molecules  It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions