Application of Capillary Electrophoresis in Optimization and Control of Fermentation and Cell Culture Analysis of Carbohydrates This method is using a direct UV detecƟon at 270 nm, by using a DAD detector and a buffer at high pH (12.6) see figure 1. General condiƟons used to obtain separaƟon showed in Figure 2. PA 800 plus, with short end injecƟon to obtain an analysis Ɵme of 4 minutes CEofix™ Carbo kit (Analis, Suarlée, Belgium), who uses an opƟ- mized method for carbohydrates analysis Samples are diluted with water, no other sample preparaƟon is required. Analysis of Anions and Organic Acids Most of the anions and organic acids do not absorb in the UV, for this reason indirect UV detecƟon is used at 230 nm by using a buffer at pH 5.6 (figure 3). General condiƟons to obtain separaƟon showed in figure 4: PA 800 plus with inverse polarity and indirect detecƟon. Anion Analysis kit (Beckman Coulter, Brea, CA, U.S.A.) Samples are diluted with a soluƟon of 5 mM NaOH. ApplicaƟon note: 12064 Figure 1: Separation of mono– and disaccharides, as well as polyols using CEofix™ Carbo kit. Figure 2: Follow‐up of glucose during fermentation of cell culture in RPMI medium, with an analysis time of 4 minutes. Figure 3: Analysis of anions and organic acids standards using Anions Analysis kit. Figure 4: Follow‐up of lactic acid during fermentation process.The analysis time is below 7 minutes. (1) Lobesh Bhattacharyya (2011). BioPharm International Supplements 24, 12-22 Introduction “The performance of fermentaƟon and cell culture, are criƟcal to the quality and consistency of producƟon of therapeuƟc products”(1). Besides parameters such as temperature, pH and dissolved oxygen, it is important to follow the nutrients and metabolites produced. Capillary electrophoresis is an excellent tool for this purpose as it combines, fast separaƟon, easy sample preparaƟon and availability of different kits and methods for the same instrument. In this “ApplicaƟon note” we will describe different methods for analysis of organic acids, caƟons, carbohydrates and proteins all using the Beckman Coulter PA 800 plus instrument (Beckman Coulter Brea, CA U.S.A.).
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Application of Capillary Electrophoresis in
Optimization and Control of Fermentation and Cell Culture
Analysis of Carbohydrates
This method is using a direct UV detec on at 270 nm, by using a DAD detector and a buffer at high pH (12.6) see figure 1. General condi ons used to obtain separa on showed in Figure 2.
PA 800 plus, with short end injec on to obtain an analysis me of 4 minutes
CEofix™ Carbo kit (Analis, Suarlée, Belgium), who uses an op -mized method for carbohydrates analysis
Samples are diluted with water, no other sample prepara on is required.
Analysis of Anions and Organic Acids
Most of the anions and organic acids do not absorb in the UV, for this reason indirect UV detec on is used at 230 nm by using a buffer at pH 5.6 (figure 3). General condi ons to obtain separa on showed in figure 4:
PA 800 plus with inverse polarity and indirect detec on.
Anion Analysis kit (Beckman Coulter, Brea, CA, U.S.A.)
Samples are diluted with a solu on of 5 mM NaOH.
Applica on note: 12064
Figure 1: Separation of mono– and disaccharides, as well as polyols using CEofix™ Carbo kit.
Figure 2: Follow‐up of glucose during fermentation of cell culture in RPMI medium, with an analysis time of 4 minutes.
Figure 3: Analysis of anions and organic acids standards using Anions Analysis kit.
Figure 4: Follow‐up of lactic acid during fermentation process.The analysis time is below 7 minutes.
(1) Lobesh Bhattacharyya (2011). BioPharm International Supplements 24, 12-22
Introduction
“The performance of fermenta on and cell culture, are cri cal to the quality and consistency of produc on of therapeu c products”(1). Besides parameters such as temperature, pH and dissolved oxygen, it is important to follow the nutrients and metabolites produced. Capillary electrophoresis is an excellent tool for this purpose as it combines, fast separa on, easy sample prepara on and availability of different kits and methods for the same instrument. In this “Applica on note” we will describe different methods for analysis of organic acids, ca ons, carbohydrates and proteins all using the Beckman Coulter PA 800 plus instrument (Beckman Coulter Brea, CA U.S.A.).
Analysis of Cations Ca ons and alipha c amines also do not absorb in the UV and an indi-rect UV detec on at 200 nm is selected using a buffer at pH 4.2. General condi ons to obtains separa on as showed in figure 7:
PA 800 plus with normal polarity and indirect detec on
Ca on Analysis kit (Beckman Coulter, Brea, CA, U.S.A.)
Samples are diluted with water
Kits may be obtained from Beckman Coulter Europe or from Beckman Coulter dealers in Europe.
Analysis of Proteins
Proteins are analyzed using CZE method with a buffer based on nitrilotri(methylphosphonic acid) or NTMP. NTMP is very UV transparent and allows buffer prepar on between pH 4 and 7.2. Monoclonal an body therapy is using monoclonal an bodies (mAb) such as Immunoglobulin G (IgG). General condi ons (figure 5):
PA 800 plus is used at 200 nm
CEofix™ NTMP is used at pH 7.2
Samples are diluted with the sample diluent solu on from the kit
For iden fica on, IgG are purified using Melon™ Gel IgG purifica-on kit (Pierce Biotechnology, Rockfort, IL USA) see figure 6.
Figure 7: Follow‐up of Ammonium during fermentation process. The analysis time is below 5 minutes.
Figure 5: IgG in cell culture supernatans, before and after concentration phase. The analysis time is 7 minutes.
Figure 6: Analysis of IgG after purification on Melon™ Gel IgG. The analysis time is 6 minutes.
Partnumber Descrip on
B20754 CEofix™ Carbo kit, contains coa ng, buffer and testmix. (200 test per kit)
B19759 Capillary for Carbo kit 25 µm I.D. x 60 cm (Package of 3)
A53537 Anion Analysis kit, contains 3 capillaries, anion coa-ng, buffer, rinse solu on, condi onner, internal
standard and test mix. (500 tests per kit)
A53540 Ca on Analysis kit, contains 3 capillaries, coa ng, buffer, rinse solu on, condi onner, internal standard and test mix (500 tests per kit).
A71142 CEofix™ NTMP kit, contains coa ng, buffer: 50 mM pH 4 and pH 7.2, (100 to 200 tests per kit)
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