20. Karst DJ, Steinebach F, Soos M, Morbidelli M

(2017) Process performance and product qual-

ity in an integrated continuous antibody pro-

duction

process.

Biotechnol

Bioeng

114

(2):298–307.

https://doi.org/10.1002/bit.

26069

21. Steinebach F, Ulmer N, Wolf M, Decker L,

Schneider V, W€alchli R et al (2017) Design

and operation of a continuous integrated

monoclonal antibody production process. Bio-

technol Prog 33(5):1303–1313. https://doi.

org/10.1002/btpr.2522

22. Burgstaller D, Krepper W, Haas J, Maszelin M,

Mohoric J, Pajnic K et al (2018) Continuous

cell flocculation for recombinant antibody har-

vesting.

J

Chem

Technol

Biotechnol

93

(7):1881–1890.

https://doi.org/10.1002/

jctb.5500

23. Rhee JI, Schu¨gerl K (1998) Continuous culti-

vation of recombinant Escherichia coli JM109

in a two-stage cascade reactor and production

of the fusion protein EcoRI::SPA. Process Bio-

chem

33(2):213–224.

https://doi.org/10.

1016/S0032-9592(97)00097-6

24. Schmideder A, Weuster-Botz D (2017) High-

performance recombinant protein production

with Escherichia coli in continuously operated

cascades of stirred-tank reactors. J Ind Micro-

biol Biotechnol 44(7):1021–1029. https://

doi.org/10.1007/s10295-017-1927-y

25. Kopp

J, Kolkmann

A-M, Veleenturf PG,

Spadiut O, Herwig C, Slouka C (2019) Boost-

ing recombinant inclusion body production—

from classical fed-batch approach to continu-

ous

cultivation.

Front

Bioeng

Biotechnol

7:297

26. Kopp J, Kittler S, Slouka C, Herwig C,

Spadiut

O,

Wurm

DJ

(2020)

Repetitive

fed-batch: a promising process mode for bio-

manufacturing with E. coli. Front Bioeng Bio-

technol 8:1312

27. Krull R, Peterat G (2016) Analysis of reaction

kinetics during chemostat cultivation of Sac-

charomyces

cerevisiae

using

a

multiphase

microreactor. Biochem Eng J 105:220–229.

https://doi.org/10.1016/j.bej.2015.08.013

28. Rosano GL, Morales ES, Ceccarelli EA (2019)

New tools for recombinant protein production

in Escherichia coli: A 5-year update. Protein Sci

28(8):1412–1422. https://doi.org/10.1002/

pro.3668

29. Slouka

C,

Kopp

J,

Hutwimmer

S,

Strahammer M, Strohmer D, Eitenberger E

et al (2018) Custom made inclusion bodies:

impact of classical process parameters and physi-

ological parameters on inclusion body quality

attributes. Microb Cell Factories 17(1):148.

https://doi.org/10.1186/s12934-018-0997-5

30. Neubauer P, Hofmann K (1994) Efficient use

of lactose for the lac promotercontrolled over-

expression of the main antigenic protein of the

foot and mouth disease virus in Escherichia coli

under

fed-batch

fermentation

conditions.

FEMS Microbiol Rev 14(1):99–102. https://

doi.org/10.1111/j.1574-6976.1994.

tb00080.x

31. Wurm DJ, Veiter L, Ulonska S, Eggenreich B,

Herwig C, Spadiut O (2016) The E. coli pET

expression system revisited—mechanistic cor-

relation between glucose and lactose uptake.

Appl

Microbiol

Biotechnol

100

(20):8721–8729

32. Metzger KFJ, Padutsch W, Pekarsky A, Kopp J,

Voloshin AM, Ku¨hnel H et al (2020) IGF1

inclusion

bodies:

a

QbD

based

process

approach for efficient USP as well as early

DSP

unit

operations.

J

Biotechnol

312:23–34.

https://doi.org/10.1016/j.

jbiotec.2020.02.014

33. Dvorak

P,

Chrast

L,

Nikel

PI,

Fedr

R,

Soucek K, Sedlackova M et al (2015) Exacer-

bation of substrate toxicity by IPTG in Escher-

ichia coli BL21(DE3) carrying a synthetic

metabolic pathway. Microb Cell Fact 14:201

34. Bru¨ckner R, Titgemeyer F (2002) Carbon

catabolite repression in bacteria: choice of the

carbon source and autoregulatory limitation of

sugar utilization. FEMS Microbiol Lett 209

(2):141–148.

https://doi.org/10.1111/j.

1574-6968.2002.tb11123.x

35. Kopp J, Slouka C, Ulonska S, Kager J, Fricke J,

Spadiut O et al (2017) Impact of Glycerol as

Carbon

Source

onto

Specific

Sugar

and

Inducer Uptake Rates and Inclusion Body Pro-

ductivity in E. coli BL21(DE3). Bioengineer-

ing

5(1):1.

https://doi.org/10.3390/

bioengineering5010001

36. Kittler S, Kopp J, Veelenturf PG, Spadiut O,

Delvigne F, Herwig C et al (2020) The Lazarus

Escherichia coli effect: recovery of productivity

on glycerol/lactose mixed feed in continuous

biomanufacturing. Front Bioeng Biotechnol

8:993. https://doi.org/10.3389/fbioe.2020.

00993

37. Wurm

DJ,

Quehenberger

J,

Mildner

J,

Eggenreich B, Slouka C, Schwaighofer A et al

(2018) Teaching an old pET new tricks: tuning

of inclusion body formation and properties by a

mixed feed system in E. coli. Appl Microbiol

Biotechnol 102(2):667–676. https://doi.org/

10.1007/s00253-017-8641-6

38. Wurm DJ, Hausjell J, Ulonska S, Herwig C,

Spadiut O (2017) Mechanistic platform knowl-

edge of concomitant sugar uptake in Escheri-

chia coli BL21(DE3) strains. Sci Rep 7:45072.

https://doi.org/10.1038/srep45072

A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3)

239