Scorpion antivenom nanobody production in Escherichia coli (Recombinant bispecific VHH)

Real-time online monitoring of bioprocesses

Industrial biotechnology (IB) has the potential to create new markets while protecting the environment. With this in mind, the EU-funded BIOINDUSTRY 4.0 project will pave the way for IB to become a major manufacturing technology. Supporting the digitalisation of IB, the project will create new services delivered by European research infrastructure. These will address several challenges, focusing on the acceleration of bioprocess development pipelines. ...

We studied the production of scorpion antivenom nanobodies in high cell density microbioreactors. The BiolectorXT (Beckman) was used to screen different production strategies within the framework of a machine-learning model optimization.

In the biopharmaceutical industry, nanobodies show promising properties for its application in serotherapy for serums targeting the most toxic components of scorpion venom. Nevertheless, the limited number of scientific publications on the production of nanobodies for antivenom productions poses a challenge. The reported yield is low (< 4 mg/L), biomass production is not reported, and the production in high cell density cultures have not been studied. Production conditions (temperature of ...

The screening of E. coli strains WK6, CH10-12 (2017), CH10-12 (2023), NbF12-10 (2017), and NbF12-10 (2023) was made in a BioLectorXT (Beckman) setting

Culture of E. coli strains WK6, CH10-12 (2017), CH10-12 (2023), NbF12-10 (2017), and NbF12-10 (2017) at 28°C in a fed-batch microbioreactor setting.

5L bioreactor culture of strain E. coli WK6 in a batch/fed-batch strategy at 37°C. Characterization of biomass growth and oxygen transfer

5L bioreactor culture of recombinant strain E. coli CH10-12 in a batch/fed-batch strategy at 37°C (BR02). Protein induction promoted when biomass reached 25 g cdw/L and at 28°C for 12 h

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 30 g cdw/L and at 29°C for 38 h

5L bioreactor culture of reference strain E. coli WK6. Protein induction promoter when biomass reached 25 g cdw/L and at 29°C for 28 h

32 fed-batch cultures at 28°C

Excel file containing the data anlysis of the BR03 culture: mass balances, specific growth rate, bioreactor volume, oxygen uptake rate (OUR)

Excel file containing the data anlysis of the BR04 culture: mass balances, specific growth rate, bioreactor volume, oxygen uptake rate (OUR)

Excel file containing the data anlysis of the BR05 culture: mass balances, specific growth rate, bioreactor volume, oxygen uptake rate (OUR)

Excel file containing the data anlysis of the BR06 culture: mass balances, specific growth rate, bioreactor volume, oxygen uptake rate (OUR)

Global results obtained from the 5 L bioreactor cultures of E. coli

5L bioreactor culture of_ E. coli _NbF12-10 at 29°C in nanobody production phase adding 1 mM IPTG to the bioreactor. Caracterization of nanobody production in glucose-limited conditions, using a constant glucose feed strategy of 4.5 g/h

5L bioreactor culture of E. coli WK6 at 29°C in biomass production phase under induction conditions adding 1 mM IPTG to the bioreactor. Caracterization of biomass production in glucose-limited conditions, using a constant glucose feed strategy of 4.5 g/h

5L bioreactor culture of E. coli NbF12-10 at 37°C in fed-batch phase. Caracterization of biomass production phase in glucose-limited conditions, using an exponential-like glucose feed strategy for a µ=0.38 h-1

5L bioreactor culture of_ E. coli_ WK6 at 37°C in fed-batch phase. Caracterization of biomass production phase in glucose-limited conditions, using an exponential-like glucose feed strategy for a µ=0.38 h-1

5L bioreactor culture of _E. coli _NbF12-10 at 37°C in batch phase. Caracterization of biomass growth

Procedure followed for the data treatment and analysis of 5L bioreactor cultures

Scorpionism is a serious public health problem in tropical areas, especially in Africa, southern India, the Middle East, and Latin America. There are about 12 species of scorpions that are dangerous to humans and all belong to the Buthidae family. In particular, venom on scorpion Androctonus australis hector is particularly toxic to humans. Serotherapy uses antibodies or fragments of antibodies to target the neurotoxins of the scorpion venom. Nanobodies, camelid-derived antibodies, are more ...

Sample taken from the 5L bioreactors, cell dry weight and HPLC data

Gas analyzer (INNOVA 1313, Lumasense Technologies) Channel 3: gas out - BR04 Channel 4: gas out - BR05 Channel 6: gas in (reference) Sampling time: 60 s Flushig time: 45 s Sampling time: 15 s

Gas analyzer (INNOVA 1313, Lumasense Technologies) Channel 3: gas out - BR02 Channel 4: gas out - BR03 Channel 6: gas in (reference) Sampling time: 60 s Flushig time: 45 s Sampling time: 15 s

5L bioreactor of E. coli NbF12-10 in a batch/fed-batch strategy at 37°C, induction promoted for 37h at 29°C MFCS BioPAT Sartorius

5L bioreactor of E. coli WK6 in a batch/fed-batch strategy at 37°C, induction promoted for 28h at 29°C MFCS BioPAT Sartorius

5L bioreactor of E. coli CH10-12 in a batch/fed-batch strategy at 37°C, induction promoted for 35h at 32°C MFCS BioPAT Sartorius