WHOLE-CELL ESCHERICHIA COLI LACTATE

BIOSENSOR FOR MONITORING

MAMMALIAN CELL CULTURES DURING
BIOPHARMACEUTICAL PRODUCTION

C. SIVAPRIYA (152BT190)
AIM:
• To monitor lactate accumulation in mammalian cell culture using E. Coli lactate biosensor.

OBJECTIVE:
• To design the whole cell E. Coli lactate biosensor.

• To monitor the lactate in mammalian cells using the designed lactate biosensor.
 INTRODUCTION

• Biopharmaceuticals are protein-based drugs, such as monoclonal antibodies and enzymes.

• During the production of biopharmaceuticals there will be the accumulation of metabolite waste products
such as, lactate.
• These lactate may decrease the cell growth and protein production.
• Current standard analytical methods for measuring lactate concentrations rely largely on hplc and
electrochemical methods, which are limited in terms of cost, sensitivity, and robustness.
• Apart from these methods, biosensor are used to overcome these limitations.
WHOLE CELL BIOSENSORS

• Research in whole-cell biosensor begun from 1970's.

• The simplest whole-cell biosensors are microorganisms, either natural or engineered used to
identify the presence of toxic molecules via a disturbance in the expression of a reporter protein.
• Whole-cell biosensors can be designed to detect any compound of interest.
• As living organism, it is self renewing that can be grown in large quantities from a single cell.
• Here, they used two genetic variations of the natural e. Coli lldprd lactate operon (fig. 1) to
construct our biosensor and tested its performance in a wide range of conditions.
 MATERIALS AND METHODS:
Application of llprd lactate gene of E. Coli by PCR

Biosensor characterization

Analysis of biosensor data and concentration estimation

 BIOSENSOR CHARACTERISATION:
• Biosensors were characterized using an aviso-gmbh theonyx robotic platform linked to a plate reader
and shaking incubator.
• A single colonies containing biosensor plasmid were inoculated in 1ml of lb medium containing
100ug/ml ampicillin and grown for 6 hr at 37 c with shaking at 250 rpm.
• Dilution of starter culture into 5ml of m9 medium and grown overnight.
• Cultures were transferred to 96 well plate format with 200ul per well.
• Using theonyx platform, the cells were allowed to attain exponential growth and 25ul of sample was
added.
• For characterization, sample with various concentration of l-lactate, d-lactate or pyruvate were mixed in
different medium ( m9 medium, cd-cho, cd-cho+ht+ 8mm glutamine, dmem, dmem with 10% calf bovine
serum).
• Od for every 10 min were measured.
 ANALYSIS OF BIOSENSOR DATA AND
CONCENTRATION ESTIMATION:

• To compare the biosensors under different conditions, the foldinduction, goodness-of-fit of the transfer
function, limit of detection, and sensitivity were calculated.
• The foldinduction was calculated as the fluorescence rate of change of samples containing 14mm
lactate divided by the fluorescence rate of change of sampleswith no added lactate.
• The limit of detection reported is the lowest lactate concentration. The goodness of fit and sensitivity
were calculated using graphpad prism.
• To calculate lactate concentrations in mammalian cell culture samples, gfp synthesis rate values from a
set of standards from the same experiment were chosen to make a standard curve and the gfp synthesis
rate values from biosensors incubated with the cell culture samples were used to interpolate
(sigmoidal, 4pl) the lactate concentrations using the graphpad prism software1.
 MAMMALIAN CELL CULTURES:

Hybridoma flask cultures

CHO-S flask cultures
GS-CHO bioreactor cultures

• The culture was inoculated in different medium.

• It was harvested on the day 16.
• These three experimental series of cell culture is used to see if the biosensor could be used to quantify
lactate metabolism in real cultures.
CROSS VALIDATION OF LACTATE CONCENTRATION:
1-5 ml of sample was taken for analysis.

Centrifuge at 800 rpm for 5 min and the supernatant was transferred to the
Fresh tube.

Store at -20 c

Concentration of ammonia, glucose, glutamine, glutamate, lactate was measured

Using bioprofile 400 analyzer.
EXPERIMENTAL WORK FLOW:
 RESULTS AND DISCUSSION:
• BIOSENSORS CAN ACCURATELY DETERMINE LACTATE CONCENTRATION IN SAMPLES
FROM A RANGE OF DIFFERENT CONDITIONS RELEVANT TO BIOPHARMACEUTICALS
INDUSTRY.

• BIOSENSOR CAN MEASURE LACTATE CONCENTRATION AS LOW AS 50UM IN M9

MEDIUM, WHICH IS APPROXIMATELY 100 FOLD INDUCTION LOWER THAN THE LOWER
LIMIT OF DETECTION OF BIOPROFILE ANALYZER.
BIOSENSOR RESPONSE TO L- LACTATE WHEN
GROWN IN CD-CHO MEDIUM
BIOSENSOR RESPONSE TO L- LACTATE SPIKED INTO
DIFFERENT MAMMALIAN CELL CULTURE MEDIA:

CD-CHO MEDIUM
CD-CHO MEDIUM WITH HT SUPPLEMENT AND 8MM GLUTAMINE
DMEM MEDIUM
DMEM MEDIUM WITH ADDED PHENOL RED
DMEM MEDIUM WITH ADDED SERUM
 CELL CULTURE EXPERIMENTS:

HYBRIDOMA CELLS GROWN IN SHAKE FLASK IN DMEM MEDIUM WITH PHENOL RED AND
SUPPLEMENTED WITH 10% SERUM AND DIFFERENT CONCENTRATION OF GLUTAMINE
 CHO- S CELL LINE EXPRESSING A FUSION PROTEIN GROWN IN BATCH CULTURE IN CD-
CHO
MEDIUM IN SHAKE FLASK
GS- CHO CELL LINE EXPRESSING MONOCLONAL ANTIBODY GROWN IN
FED BATCH REACTOR WITH CD-CHO MEDIUM
 CONCLUSION:

• We have created a functional whole-cell lactate biosensor based on the E. Coli lldprd operon.

• They have demonstrated that this biosensor can be used for monitoring lactate in complex
industrial contexts, specifically mammalian cell cultures producing a recombinant protein, with
high accuracy and a lower limit of detection than the industry standard method.
THANK YOU........