Presentation Information
[P04-482]Process Parameter Influence on Inclusion Body Formation in Recombinant scfv Production by E. coli
○Ankita Rao Kokolu1, Dr Guhan Jayaraman1 (1. Indian Institute of Technology, Madras (India))
Keywords:
Inclusion Bodies (IB),single chain variable fragment (scFv),Quality attributes
Recombinant single chain variable fragment (scFv) antibodies hold significant potential for therapeutic and diagnostic application due to their specificity and versatility. But when produced in cost effective hosts like E. coli, they are often expressed during heterologous expression as inclusion bodies (IB). IB’s are dense aggregates of misfolded proteins but increasing evidence indicate that they retain their secondary and tertiary structural characteristics during refolding. Literature further suggests that upstream process variables shape IB properties, yet systematic frameworks linking production strategy to IB quality attributes remain limited.
In this study, we investigate how process variables modulate key IB quality attributes – titer, size and purity, using an anti-cd99 scFv expressed in E. coli under batch and fed batch conditions. IB quality was characterised by dynamic light scattering (DLS) to assess particle size distribution and heterogeneity, Far-UV circular dichroism (CD) or FTIR to evaluate secondary structural retention in solubilised IB. Purity and oligomeric distribution were evaluated using Size exclusion chromatography- HPLC with SDS-PAGE. Intrinsic fluorescence spectroscopy provided additional insight into tertiary packing around aromatic residue exposure under different processing condition.
Glucose based fed batch led to >10g/L acetate accumulation which resulted in reduced IB productivity whereas shifting to xylose as an alternate carbon source not only yielded a 1.7-fold increase in solubilised protein but a 3-fold increase in Yp/x highlighting the impact of overflow metabolism on expression performance. Ongoing efforts are examining how inclusion body focused process optimization intersects with phage susceptibility. Together, these establish a process framework for IB formation in scfv production, future emphasis would be on systematically investigating how aggregation outcomes are determined not solely by protein sequence but also by cultivation strategy, to enable scalable workflows that balance productivity with downstream recoverability.
In this study, we investigate how process variables modulate key IB quality attributes – titer, size and purity, using an anti-cd99 scFv expressed in E. coli under batch and fed batch conditions. IB quality was characterised by dynamic light scattering (DLS) to assess particle size distribution and heterogeneity, Far-UV circular dichroism (CD) or FTIR to evaluate secondary structural retention in solubilised IB. Purity and oligomeric distribution were evaluated using Size exclusion chromatography- HPLC with SDS-PAGE. Intrinsic fluorescence spectroscopy provided additional insight into tertiary packing around aromatic residue exposure under different processing condition.
Glucose based fed batch led to >10g/L acetate accumulation which resulted in reduced IB productivity whereas shifting to xylose as an alternate carbon source not only yielded a 1.7-fold increase in solubilised protein but a 3-fold increase in Yp/x highlighting the impact of overflow metabolism on expression performance. Ongoing efforts are examining how inclusion body focused process optimization intersects with phage susceptibility. Together, these establish a process framework for IB formation in scfv production, future emphasis would be on systematically investigating how aggregation outcomes are determined not solely by protein sequence but also by cultivation strategy, to enable scalable workflows that balance productivity with downstream recoverability.
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