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Baganz, Frank

Cuccui, Jon

Kay, Emily

Mukhopadhyay, Tarit

Patel, Neha

Sulu, Michael

English

Engineering Conferences International

Acknowledgments Background• Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease inchildren resulting in pneumonia and meningitis.• Introduction of glycoconjugate vaccines led to a significant reduction in invasive bacterialdiseases in young children worldwide.• Current commercial glycoconjugate vaccine for immunisation is Prevnar-13 but cost perdose is high due to complex and long manufacturing process (Fig. 1).• Protein Glycan Coupling Technology (PGCT) is promising alternative to produceglycoconjugate vaccines intracellularly in E. coli [1, 2] (Fig. 2).Materials and MethodsFed-batch fermentation in defined mediumReferencesOptimisation of fermentation conditions using DoE ConclusionsImpact of pglB location and a different acceptor protein on glycoconjugate productionFigure 1: Overview of the chemical conjugation method of glycoconjugate vaccine production.Figure 2: Schematic of PGCT system to produce glycoconjugate recombinantly in E. coli. 1) The oligosaccharide is assembled on a lipid core undecaprenyl pyrophosphate (Und-PP) on the cytoplasmic face to form a lipid linked oligosaccharide (LLO), 2) which is flipped into the periplasm by a flippase. 3) A polymerase will then add repeat units of the oligosaccharide to assemble the glycan with multiple repeat units. 4) The oligosaccharyltransferase PglB will recognise the reducing end sugar of the first monosaccharide of the oligosaccharide and transfer the glycan onto the glycotag on the acceptor protein to form the final glycoconjugate product in the periplasm. : Strain NumberStrain and plasmids componentsDescription pglB LocationAcceptor ProteinSource1 W311Bpb4 pEXT20 (galE)pEXT21 (piuA)Production of ST4- PiuAGlycoconjugateChromosome PiuA Reglinski et al.  (2018)2 W3110pb4 pEXT200 (galE+pglB pEXT21 (piuA)Production of ST4- PiuAGlycoconjugatePlasmid PiuA This Study3 W311Bpb4 pEXT20 (galE+pglB) pEXT21 (exoA)Production of ST4- ExoAGlycoconjugateChromosome ExoA This Study4 W3110pb4 pEXT20 (galE+pglB) pEXT21 (exoA)Production of ST4- ExoAGlycoconjugatePlasmid ExoA This StudyFermentation ModeMedia Inoculation conditionStarting Working VolumePre-Induction Feed ConditionSpecific Growth Rate value for Exponential FeedPost-Induction Feed ConditionInduction ConditionsBatch SSOB Cells inoculated into bioreactor to an inoculation OD600between 0.3-0.5200 mL Not applicableNot applicable Not applicable1 mM IPTG,  4 mM MnCl2 induction at inoculationFed-Batch SM6Gc30 g/L  or 100 g/L   Glycerol in batch phaseCells inoculated into bioreactor to an inoculation OD600between 0.3-0.5200 mL Not applicableNot applicable 80% (w/w) Glycerol fed at a rate of 1.6 mL/L/h*Feed initiated at DOT spike1 mM IPTG, 4 mM MnCl2 induction at DOT Spike4 mM IPTG, 4 mM MnCl2 induction at DOT Spike4 mM IPTG, 8mM MnCl2 induction at DOT SpikeFed-Batch with exponential feedSM6Gc30  g/L   Glycerol in batch phaseCells inoculated into bioreactor to an inoculation OD600 of approximately 0.5140 mL Exponential feed  initiated at DOT spike maintaining specific growth rate of cells0.06 h-1 or 0.12 h-1 in preliminary experiments.  0.07 h-1, 0.085 h-1 or 0.1 h-1 for fermentations required for DoE study.80% (w/w) Glycerol fed at a rate of 1.6  mL/L/h. Feed initiated at OD600  138 +/- 9.61 mM IPTG 4 mM MnCl2 induction at OD600  138 +/- 9.6Table 1: E. coli strains used for glycoconjugate production.Table 2: Summary of fermentation protocols used in bioreactors.• Develop a fed-batch fermentation protocol in a defined medium.• Investigate the impact of IPTG concentration and MnCl2 supplementation on glycoconjugateproduction in fed-batch fermentations using Strain 1 (Table 1, 2).Figure 3: Fermentation traces for fed-batch fermentations. (A) fermentation trace for a condition with 30 g/L glycerol in the batch phase and (B) a fermentation trace for 100 g/L glycerol in the batch phase. Arrow indicates point of DOT spike, start of feed of 800 g/L glycerol at a rate of 1.6 mL/L/h and point of induction with 1mM IPTG and supplementation with 4mM MnCl2.Figure 4: Immunoblot (A) and densitometry analysis (B) of endpoint samples taken from various fed-batch protocols 28 h post-induction. All samples were OD600 matched. Densitometry analysis was performed on glycoconjugate bands. The boxes indicate the bands which were included in this analysis.  • Glycoconjugate production in normalised samples highest in fermentation with 30g/L glycerol. • Increasing IPTG led to slightly higher glycoconjugate production using 100 g/L glycerol.  • Construct three new strains which either express PglB on a plasmid or have a differentacceptor protein present (Figure 5).• Compare the performance of the strains in terms of biomass and glycoconjugate productionin fed-batch fermentations with defined medium.Figure 6: Immunoblot (A) and densitometry analysis (B) of endpoint samples taken from fed-batch fermentations 28 h post-induction. All samples were OD600 matched. Densitometry analysis was performed on glycoconjugate bands. Figure 5: Schematic of glycoconjugate producing strains tested(Table 1). • Strains with ExoA as an acceptor protein (Strains 3 and 4) have higher glycoconjugate production.• The glycoconjugate production of a chromosomal versus a plasmidic strain is similar with ExoA as the acceptor protein.• Investigate the effect of pre-induction growth rate, post-induction temperature and post-induction feed rate on cell biomass and glycoconjugate production using Strain 3.Figure 7: Schematic of central composite design outlining the factors and levels chosen and the responses that were measured. In total 18 different conditions including 4 centre points were run (Table 2). Figure 8: Scaled densitometry signal for glycoconjugate. Bars in green are runs which had a signal higher than the fed-batch benchmark (FFB) sample. Figure 9: Response surface model plots for OD600 (A), Dry cell weight (B) and scaled densitometry signal (C). • DOE study has improved cell biomass yield at harvest between 3-7.3 fold compared to a fed-batch benchmark condition. • Densitometry signal compared to a benchmark sample improved in only three conditions when running normalised samples on immunoblots (Fig. 8)• Post-induction feed rate and temperature have a statistically significant impact on biomass at harvest and scaled densitometry signal. • First study to demonstrate production of a serotype 4 pneumococcal glycoconjugate using PGCT in fed-batch fermentation with defined medium. • The choice of acceptor protein has a considerable impact on glycoconjugate production. • Optimisation of fermentation process variables can further increase cell biomass yield and glycoconjugate production. Towards a platform process for the manufacture of glycoconjugate vaccines for pneumococcal disease Neha Patel1, Emily Kay2, Jon Cuccui2, Tarit Mukhopadhyay1,3, Michael Sulu1, Frank Baganz1*1 Department of Biochemical Engineering University College London, U.K.2 London School of Hygiene and Tropical Medicine, U.K.3 Merk Research Laboratories, USA*f.baganz@ucl.ac.uk1. Cuccui J, et al. (2013) Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open Biol.; 3(5):130002.2.  Reglinski M, et al. (2018) A recombinant conjugated pneumococcal vaccine that protects against murine infections with a similar efficacy to Prevnar-13. npj Vaccines 3.ABABA B C

Towards a platform process for the manufacture of glycoconjugate vaccines for pneumococcal disease

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Towards a platform process for the manufacture of glycoconjugate vaccines for pneumococcal disease

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Engineering Conferences International