The amino acid compositions of cell culture and fermentation broths can be determined by HPAEC-PAD without sample derivatization
(13–15). However, the presence of carbohydrates and alditols in the media may interfere. By optimizing the elution method,
Genzel et al. (13) reported that the amino acids were well resolved and also resolved from the predominant carbohydrate, glucose,
in GMEM media used in the production of inactivated influenza vaccines (see Figure 5). The authors reported good precision,
accuracy, linear range, and low quantitation limit for each amino acid. Similar results were also reported for the supernatants
of YPD broth, LB broth, Minimum Essential Eagle's Medium and a serum-free, protein-free hybridoma medium (14, 15).
Figure 5: Amino acid analysis of (1) a standard amino acid mixture containing 277.5 µM glucose;(2) GMEM medium; (3) GMEM medium
+ 1% (v/v) peptone; (4) GMEM medium+10% (v/v) FCS + 1% (v/v) peptone. Norleucine was added as the internal standard. Column:
AminoPac PA10; elution: as per the procedure described in Ref. 16. (Reprinted from Ref. 13 with permission from Elsevier)
Selective media are used for the growth of select microorganisms. For example, if a microorganism is resistant to a certain
antibiotic, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance,
from growing. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully
transfected with a plasmid carrying the neomycin resistance gene as a marker. Generally, neomycin is assayed following the
procedure described in USP–NF General Chapter <81>, Antibiotics—Microbial Assay (17). However, the procedure is tedious and labor intensive, has poor precision,
and requires days to complete. Neomycin is an aminoglycoside containing a carbohydrate moiety and, like other carbohydrates,
can be assayed using HPAEC–PAD. Figure 6 shows the HPAEC–PAD chromatograms of two selective media containing neomycin. The
media also contain glucose, sucrose, and amino acids at concentrations 100-1000 fold higher than that of neomycin. These components
also show peaks in HPAEC–PAD. Thus, the resolution of neomycin was a challenge, which was accomplished using a CarboPac PA1
column and a linear elution gradient from 1.0 mM to 3.0 mM NaOH over 25 min.
Figure 6: HPAEC-PAD chromatograms of a neomycin standard and two selective culture media containing neomycin. Column: CarboPac
PA1; flow rate: 0.5 mL/min. The effluent from the column was mixed with 100 mM NaOH before entering into the detector for
Monitoring nutrients and metabolites in cell culture media and fermentation broth provides critical pieces of information
on their roles in fermentation, and in optimizing, characterizing, and controlling the process to produce safe and efficacious
therapeutic products in a reproducible manner. The results presented in this review show that the unique selectivity and sensitivity
of IC with ED permit determination of fermentation broth components. This is particularly important for those components which
do not have suitable chromophores for detection by absorption measurement. The sample preparation involves simple filtration
and dilution of fermentation broth with water. No pre- or postcolumn derivatization is necessary for detection. In addition,
the technique provides the ability to analyze the desired components selectively in the presence of others, e.g., amino acids
in the presence of large excess of carbohydrates or inorganic cations and anions in the presence of amino acids. These features
make IC with ED an effective technique in analysis of fermentation broths in a simple and cost effective manner.
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*The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and
should not be construed to represent any Agency determination or policy.