Application of Ion Chromatography with Electrochemical Detection in Optimization and Control of Fermentation and Cell Culture - An overview of applications in the analysis of nutrients and


Application of Ion Chromatography with Electrochemical Detection in Optimization and Control of Fermentation and Cell Culture
An overview of applications in the analysis of nutrients and metabolites.

BioPharm International Supplements
Volume 24, Issue 5, pp. s12-s20

Cations and anions

Fermentation broths typically have several cations and anions, including amino, carboxylic and other organic acids, as well as amines and other organic bases. However, concentrations of inorganic cations and anions are significantly higher than their organic counterparts. Thus, inorganic ions show significant peaks by IC when fermentation broths are diluted 1000-fold or more, while their organic counterparts are reduced to undetectable levels. This provides necessary selectivity for analysis.

The first report of the determination of cations and anions using IC to follow a fermentation process of methanotrophic bacteria was published in 1992 (7). Using OmniPac PAX-500 anion-exchange column and SCD, the authors followed several anions over the course of fermentation and found acetate to accumulate when natural gas was used as the substrate, which inhibits the growth of the methanotrophic bacteria. Mono- and divalent cations were monitored by SCD using IonPac CS10 cation-exchange column eluted isocratically with 40 mM HCl–4 mM DL-2,3-diaminopropionic acid (DAP) as the eluent. The results indicated that the concentration of Mg2 + must be above 60 ppb to maintain optimum growth of the bacteria.

Figure 1: A time course of analysis of cations in an insect cell culture using a complex medium with glucose and sucrose as the primary carbon sources. Peaks: 1=sodium, 2=ammonium, 3=potassium, 4=magnesium, and 5=calcium. Column: IonPac CS12; elution: 20 mM MSA, isocratic, 1.0 mL/min. (Reprinted from Ref. 9 with permission from Elsevier)
Robinette et al. (8, 9) reported quantitation of eight mono- and divalent cations in fermentation broths for recombinant and pathogenic microorganisms, as well as in mammalian and insect cell cultures in complex and defined media. The authors employed IonPac CS10 and CS12 cation-exchange columns eluted isocratically with 20 mM HCl – 4 mM DAP and 20 mM methanesulfonic acid (MSA), respectively. The cations are detected by SCD. The sample preparation involves simple 1000–2500 fold dilution of filtered broth with water. The reports indicate good precision and accuracy, and a wide linear range for each cation, with detection limits ≤1 g/mL.

Figure 2: Resolution of carbohydrates, alditols, alcohols, and glycols commonly found in fermentation broths by (A) CarboPac MA1 column and (B) CarboPac PA1 column. (Reprinted from Ref. 12 with permission from Dionex)
Figure 1 shows a time course of an insect cell culture using a complex medium. The chromatograms show that the levels of sodium, potassium, magnesium, and calcium remained virtually unchanged throughout the fermentation, while ammonium concentration increased approximately 2-fold. Similar investigations were reported for S. cerevisiae (eukaryotic) grown in a chemically defined medium, and E.coli/ (prokaryotic) and a mammalian cell culture in complex media containing glucose as the primary carbon source.

Figure 3: A time course of analysis of carbohydrates in H. influenzae cultivation using complex medium with glucose as the major carbon source. Peaks: 1=glucose, 2=fructose, 3=ribose; 4=maltose. H=hours. Elution: 150 mM NaOH, isocratic, 1.0 mL/min. (Reprinted from Ref. 11 with permission from Elsevier)
Aliphatic carboxylates (e.g., acetate, lactate, pyruvate) are metabolites that often reduce fermentation yields. In an Application Note (10), Dionex reported the analysis of aliphatic carboxylates and inorganic anions in the fermentation broths of S. cerevisiae in YPD medium and E.coli/ in LB medium using IonPac AS11-HC column eluted with 1-60 mM NaOH and SCD. The results show well resolved peaks with good precision and accuracy, wide linear range and low detection limit for each anion.

Carbohydrates and alcohols

Figure 4: Chromatograms of the analysis of S. cerevisiae fermentation broth using CarboPac MA1; (A) 0 h and (B) 24 h. (Reprinted from Ref. 12 with permission from Dionex)
Carbohydrates are carbon sources essential for cell growth and product synthesis, while alditols and alcohols are metabolites. High Performance Anion-Exchange Chromatography (HPAEC) coupled with PAD (HPAEC–PAD) or Integrated PAD (IPAD), which is an improved PAD application, is the most widely used chromatographic technique for the analysis of carbohydrates and alditols (3–6). The CarboPac MA1 column has the ability to analyze mono- and disaccharides, alditols, and alcohols present in fermentation broths (see Figure 2A) in a single run. However, the chromatography takes as long as 60 min for each run and uses a high concentration of NaOH as the mobile phase. The CarboPac PA series of columns (e.g., PA1, PA10, PA20) take significantly less time and use low NaOH concentration. But alcohols and alditols are eluted early and are poorly resolved (Figure 2B). Therefore, the PA series columns are better suited for mono- and disaccharide analysis. Thus, the choice of column depends on the analytes to be monitored in the media. Herber and his colleagues (8, 11) used a CarboPac PA1 column to monitor microbial fermentations using chemically defined and complex media. The samples were diluted 50 fold with water and eluted from the column with NaOH (isocratic and gradient) to monitor ethanol, glycerol, galactose, glucose, mannose, fructose, raffinose, ribose, and lactose. Carboxylic acids and inorganic ions are transparent to PAD. Most of the other media components were below the detection limit at this dilution. Indeed, only proline, arginine and lysine exhibited any noticeable detector response (see later for the detection of amino acids by PAD). Figure 3 shows a time-course study of carbohydrates in a H. influenzae fermentation in a complex medium with glucose as the major carbon source. Figure 4 shows the chromatograms of the analysis of S. cerevisiae fermentation broth using CarboPac MA1 (12). Glucose is the most predominant component of the media at 0 h. At the end of fermentation, no glucose was detected and the predominant components are the metabolites.

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