1. Application of Capillary Electrophoresis in follow-up of Fermentation and Cell-Culture
François de l’Escaille, Jean-Bernard Falmagne
ANALIS s.a. • R&D Diag • Zoning Industriel de Rhisnes • rue de Néverlée, 11 • 5020 Suarlée (Namur) • Belgium • Tel: + 32 81 25 50 50 • Fax: + 32 81 23 12 37 • ceofix@analis.be • http://www.analis.com/ceofix
1. Introduction :
“The performance of fermentation and cell culture, are critical to the quality and consistency 2.4. Analysis of Proteins 3.3. Analysis of Cations
of production of therapeutic products” (1). Besides parameters such as temperature, pH and Proteins are analyzed using CZE method with a Beside the presence of Sodium, Potassium,
dissolved oxygen, it is important to follow the nutrients and metabolites produced. Capillary buffer based on nitrilo-tri (methylphosphonic acid) Magnesium and Calcium we can easily follow
electrophoresis is an excellent tool for this purpose as it combines, fast separation, easy sample or NTMP. NTMP is very UV transparent and allows the evolution of Ammonium, which is a result
preparation and availability of different kits and methods for the same instrument. buffer preparation between pH 4 and 7.2. of the decomposition of amino acids and
In this “poster” we will describe different methods for analysis of carbohydrates, organic acids, General conditions (Figure 4): influence the cell growth (Figure 8).
cations, and proteins all using the Beckman Coulter PA 800 plus instrument (Beckman Coulter
- PA 800 plus is used at 200 nm
Brea, CA U.S.A.). 3.4. Analysis of Proteins and production
- CEofix™ NTMP is used at pH 7.2
mAb IgG and IgM in Cell Supernatants
2. Methods : - Samples are diluted with the sample
IgG is followed in the cell culture supernatant
diluent solution from the kit
2.1. Analysis of Carbohydrates Fig. 4 : Analysis of human serum diluted 1/40 using and analysed, before and after process Fig. 7 : Evolution of the lactic acid concentration during cell-
- For identification, IgG are purified using Melon™ the CEofix™ NTMP kit before and after purification on
concentration. Furthermore the presence of culture on RPMI medium (left) and on IMDM medium (right).
This method is using a direct UV detection at 270 Gel IgG purification kit (Pierce Biotechnology, Melon™ Gel IgG.
nm, by using a DAD detector and a buffer at high pH IgG is confirmed by the purification of the
Rockford, IL USA) see figure 5. supernatant using Melon™ Gel IgG (Figure 9).
(12.6) see Figure 1.
General conditions used to obtain separation
showed in results:
3. Results :
- PA 800 plus, with short end injection to obtain We obtained samples from a company making
Fig. 1 : Analysis of test mix of carbohydrate and polyols
an analysis time of 4 minutes using the CEofix™ Carbo kit. mAb for diagnostic purpose. mAb were grown
- CEofix™ Carbo kit (Analis, Suarlée, Belgium), in RPMI media and in IMDM media. Samples
who uses an optimized method for carbohydrates were collected on day 0, 1, 2, 3, 4 and day
analysis 7, but also the supernatant at the end of the
fermentation and after process concentration. Fig. 9 : Analysis of IgG from cell culture supernatant, left before
- Samples are diluted with water; no other sample and right after purification on Melon™ Gel IgG. In blue raw cell
preparation is required. Methods were adapted to obtain short culture supernatant IgG at 27µg/mL and in red after process Fig. 8 : Evolution of the ammonium concentration during cell-
analysis time, such as less than 4 minutes for Fig. 5 : Evolution of the glucose concentration during cell-culture concentration IgG at 150 µg/mL. culture on RPMI medium (left) and on IMDM medium (right).
on RPMI medium (left) and on IMDM medium (right).
2.2. Analysis of Anions and Organic Acids carbohydrates, 7 minutes for organic acids,
Most of the anions and organic acids do not absorb 5 minutes for cations and 6 minutes for proteins
in the UV, for this reason indirect UV detection is used Conclusion :
at 230 nm by using a buffer at pH 5.6 see Figure 2. Fig. 2 : Analysis of a test mix of anions and organic acids 3.1. Analysis of Carbohydrates Capillary Electrophoresis Using existing methods and kits and with a minimum of sample
at different concentrations using the Anion Analysis kit.
General conditions to obtain separation showed in Glucose or other carbohydrates are necessary for preparation, mostly dilution of the sample; CE is able to follow several parameters during
results: cell-growth. With this method we can follow the fermentation process. It can not only identify the analyte of interest, but can monitor other
- PA 800 plus with reverse polarity and indirect evolution for example of glucose (Figure 5). When parameters to trace when a problem occur.
detection. dealing with sorbitol pathway, instead of a glycolysis
- Anion Analysis kit (Beckman Coulter, Brea, CA, U.S.A.) pathway we can also follow sorbitol and fructose.
- Samples are diluted with a solution of 5 mM
3.2. Analysis of Organic Acids
NaOH.
With the Anion analysis kit it is possible to identify
Fig. 6 : RPMI medium spiked with organic acid test mix
2.3. Analysis of Cations anions such as phosphate, sulphate, chloride and
to identify position of analytes.
Cations and aliphatic amines also do not absorb in Fig. 3 : Analysis of a test mix of cations using the Cation to follow different organic acids in the medium
the UV and an indirect UV detection at 200 nm is Analysis kit (see figure 6): such as lactate, acetate, pyruvate,
selected using a buffer at pH 4.2. formate, glutamate (not showed). Bibliographie:
(1) Lobesh Bhattacharyya (2011). BioPharm International Supplements 24, 12-22
General conditions to obtain separation as showed in Figure 3: When following the formation one can see the
evolution of lactic acid formation over time (Figure 7).
R&D-Poster-CePharm-Fermentation&Cell-culture-0912
- PA 800 plus with normal polarity and indirect detection
- Cation Analysis kit (Beckman Coulter, Brea, CA, U.S.A.)
- Samples are diluted with water