Speciality Chemicals Magazine MAR / APR 2024 | Page 34

PHARMACEUTICALS

Fluorescence detection of resultant viral particles was facilitated by the EVOS M7000 imaging system at 4x magnification . Furthermore , viral titers were precisely determined utilising the Stunner system with UV detection at wavelengths of 254 and 280 nm .

Experimental results

The study meticulously investigated the kinetics of complexation reactions between plasmid DNA and transfection reagents , probing various ratios over a 60-minute timeframe . Gel electrophoresis coupled with dynamic light scattering ( DLS ) analyses provided critical insights into the complex formation process .

At a FectorVIR AAV : DNA ratio of 0.1:1 , incomplete complexation persisted even after 60 minutes , while at a ratio of 1:1 stability was achieved throughout the duration . Notably , Product 1 exhibited full DNA binding even at the lowest tested ratio of 0.1:1 , showcasing its exceptional efficiency . DLS analysis revealed the temporal evolution of complex sizes , with smaller particles observed for DNA and reagent individually compared to their complexes .

Dose-response studies further confirmed these findings , illustrating varied complexation efficiencies among the different transfection reagents . Interestingly , both FectoVIR AAV and Product 1 displayed optimal performance at low ratios , contrasting with the behaviour of Products 2 and 3 .

Moreover , particle size distributions elucidated optimal conditions for efficient transport across cell membranes . Additionally , larger-scale evaluation shed light on nuanced differences in transfection efficiency and particle composition among the reagents . Notably , FectoVIR AAV at a

0.6:1 ratio yielded superior virus titers and distinct particle distributions compared to other reagents tested .

These findings underscore the intricate interplay between complexation parameters and transfection outcomes , offering valuable insights for optimising protocols in AAV production . Further analysis using advanced techniques , such as next-generation sequencing , is recommended to unravel the underlying molecular mechanisms driving these observations .

Key takeaways

Preparation of complexation reagents is a key first step in transient transfection for the production of viral vectors used in gene and genemodified cell therapies and viralvectored vaccines . The experiments we conducted clearly demonstrate the importance of understanding the CPPs of the complexation process . The complexation ( stirring ) time , the ratio of plasmid DNA to transfection reagent and the choice of reagent all impact transfection efficiency and overall performance .

These factors are important because it is essential for DNA-reagent complexes to remain stable , not only during initial mixing but also while the complexation mixture is added to the bioreactor . At laboratory scale , time is not a factor because volumes are small and transfer times are short .

At commercial scale , however , much larger volumes are involved . These must be mixed and transferred carefully and slowly to avoid shear forces that can damage the sensitive complexes . Generally , these must be stable for up to 60 minutes or even more .

In general , the series of studies performed with FectoVIR AAV , Product

1 , 2 , and 3 reagents contributed to a greater understanding of the initial complexation step . The main conclusions are :

• The choice of transfection reagent has a direct impact on the performance of the transfection step

• The complexation step may be influenced by different reagents , some of which provide better transfection efficiencies

• The DNA-transfection reagent ratio is a critical process parameter

• Complexation time has an impact on the transfection efficiency for FectoVIR AAV and the Product 2 and 3 reagents

• The average particle sizes of DNA-reagent complexes formed under optimum conditions are much larger than the 300-500-nm range reported in the literature as being ideal for transport across cell membranes in vivo Greater understanding of the process parameters critical to complexation enables the development of optimum , robust , efficient , scalable transient transfection processes for the generation of viral vectors . This process knowledge is applicable across different AAV vectors , and potentially other vector types as well . ●

* - Also contributing to this article : Dr Paul Kroeger , Dr Khan Umaer , Srivanya Tummala , Margie Campbell & Dr Brian Tomkowicz , all of SK Pharmteco

** - FectorVIR is a registered trademark of Polyplus

Dr Brian Tomkowicz

SENIOR DIRECTOR

SK PHARMTECO k + 1 866 274 4009 J btomkowicz @ cfbm . com j www . skpharmteco . com /

34 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981