Speciality Chemicals Magazine MAR / APR 2024 | Page 33

PHARMACEUTICALS

ITR

Figure 2 – Steps involved in generating recombinant AAV by triple transfection

AmpR

ITR vector

Ad helper vector

REP CAP

ITR

However , even for advanced transfection reagents , complexes formed with plasmid DNA tend to be unstable and shear-sensitive , limiting the stirring speed for mixing , the rate at which they can be transferred from the mixing tank to the bioreactor , and the time they remain available for transfection into HEK293 cells . Optimising complexation processes is therefore essential .

Building know-how

Traditionally , building know-how has involved investigating process parameters individually . More recently , quality-by-design ( QbD ) approaches that leverage design of experiment ( DoE ) studies have become increasingly common .

A QbD strategy makes it possible to identify the critical process parameters ( CPPs ) that directly impact viral vector critical quality attributes . 6 , 7 , 8 Even DoE studies that explore only a few key parameters simultaneously can lead to more robust , efficient and productive transient transfection processes . 9 , 10

Three main parameters can impact the complexation step : the complexation reagent , the ratio of transfection reagent to plasmid DNA and complexation time . These influence the size of the complexes generated , which has been linked with transfection efficiency .

The ideal particle size for optimal transport across cell membranes is 400 – 500 nm . 11 Thus it can be assumed that transporting DNA-reagent complexes with larger macromolecular structures would be inefficient .

The transfection reagent to plasmid ratio will influence the kinetics of complexation and thus complexation efficiency . Using smaller quantities of plasmids and / or transfection reagent to achieve optimum transfection efficiency would provide a mechanism for reducing the cost of goods .

Complexation time , while not an issue at R & D scale , can become significant when scaling a transient transfection reaction from 50L scale to 200-500L and beyond . The stability of the plasmid DNA-transfection reagent complexes becomes a critical parameter , as the transfer of large volumes of complex mixture generally takes 30-60 minutes . During that time , degradative shear forces should be reduced in order to avoid inefficiencies in transfection .

Finally , the choice of transfection reagent can impact the efficiency of the complexation step and ultimately the productivity of the transfection process . Reagents based on lipofectamine , PEI and lipid-polymer complexes are available . PEI is generally considered the gold standard transfection reagent .

FectorVIR * AAV is designed specifically for suspension-based transient transfection of AAV vectors . It was tested against products 1 , 2 and 3 , which are cationic lipid polymer reagents for research-scale transient transfection in various mammalian cells ( including both HEK293 and Chinese hamster ovary ).

To obtain a greater understanding of how these three parameters impact the complexation process , a series of experiments was conducted . Complexation processes were performed for different periods of time using a range of plasmid ( for AAV generation ) -reagent ratios and the four different transfection reagents .

A full-length description of the study methods and experimental results is available online . 12 This covers the impact of the DNA-complexation reagent ratio on complex formation and of complexation parameters on transfection efficiency .

Study methods

Complexation reactions followed manufacturer instructions , executed within test tubes at ambient room temperature . The sole modifications involved adjusting the ratio of plasmid DNA to transfection reagent and varying complexation time .

Each experiment included a crucial control comprising DNA alone . Kinetics were scrutinised by analysing samples at intervals of 0 , 5 , 15 , 30 and 60 minutes . Meanwhile , dose-response studies entailed exposing reactions to ratios ranging from 0.1:1 to 3.0:1 for 30 minutes .

Gel electrophoresis , employing SYBR Gold staining , elucidated levels of free DNA and complexation efficiency . Isothermal stability over 60 minutes was assessed via dynamic light scattering using the Uncle system ( Unchained Labs ), while quantification of DNA-transfection reagent complex formation employed an Aura particle size analyser .

Triple transient transfections meticulously evaluated different complexation mixtures , media formulations , mixing durations and DNA-complexation reagent ratios . Within this context , AAV2 or AAV8 vectors were synthesised utilising the pAAV-CMV-green fluorescent protein ( GFP ) packaging plasmid , inducing GFP expression .

MAR / APR 2024 SPECCHEMONLINE . COM

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