Drugs, biological therapeutics, and vaccines manufactured under cGMP must be controlled at all stages of the production process.
The final release testing is not in itself sufficient to guarantee quality, safety, purity and efficacy of the final product
(1). It is necessary to ensure that the manufacturing operations perform as intended within the established specifications.
This implies that any given process is well characterized, adequately controlled, and reproducible. Fermentations are complex
processes used in the manufacture of recombinant proteins and peptides, vaccines, and other therapeutic products. The performance
of fermentation (and cell culture) processes are critical to the quality and consistency of production of the therapeutic
products and are influenced by parameters, such as pH, temperature, dissolved oxygen, as well as nutrients and other components
necessary for cell growth, and metabolites. The nutrients include carbohydrates, amino acids, and other organic compounds,
which act as carbon sources essential for cell growth and product synthesis. Inorganic cations (metal ions) also play critical
roles for optimum activities of enzymes and in cell growth. The metabolic products sometimes act to inhibit the product synthesis
and affect the desired yield. Thus, measurements of nutrients and metabolites in fermentation broths (and culture media) are
important for understanding the roles they play and microbial physiology, as well as for the development, optimization, characterization,
and control of the process. Such measurements also permit improvement of the process performance by providing the ability
to determine the need for supplementation, as well as to the development of defined media to replace complex media formulations.
Most of the nutrients and metabolites, including inorganic ions, carbohydrates, alditols, alcohols, and aliphatic carboxylic
and amino acids are ionic or polar in nature, and do not have chromophores necessary for analysis by absorption measurements.
Ion chromatography (IC) with electrochemical detection (ED) presents a suitable technique for the determination of these components.
The technique usually requires no sample preparation other than dilution with water. Other chromatographic methods often require
complex pre- or postcolumn derivatization for the detection of analytes.
This article will provide a brief overview of IC and ED, as well as their applications in the analysis of nutrients and metabolites
in fermentation broths and neomycin in selective culture media.
IC is a form of high-performance liquid chromatography (HPLC), which involves separation based on ionic interactions among
ionic or polar analytes, ions present in the mobile phase (eluent), and ionic functional groups on chromatographic support
(stationary phase). This can lead to two distinct mechanisms of separation based on the type of interactions—(a) ion exchange
in which the analyte ions are adsorbed to and desorbed from the stationary phases repeatedly leading to separation, and (b)
ion exclusion due to repulsion between similarly charged analyte ions and the ions on the chromatographic support. Separation
based on ion exchange has been the predominant application of IC to-date.
In ion-exchange chromatography, ionic functional groups on the chromatographic support have charges opposite to those of the
analyte ions. That is, a column for separation of cations, a cation-exchange column, contains negative ions. This technique
is widely used in the analysis of cations, including metal ions, anions, mono- and oligosaccharides, alditols (sugar alcohols),
aminoglycosides (antibiotics), amino acids, peptides, organic acids, amines, alcohols, glycols, thiols, and other polar molecules.
Ion-exclusion chromatography uses strong cation- or anion-exchange columns to separate ionic, polar, weakly polar, and apolar
analytes. The ionic analytes experience strong repulsion from similarly charged functional groups on the chromatographic support
and are eluted first, with little or no separation. An apolar analyte experiences no repulsion and is, therefore, eluted late
from the column. Polar analytes experience less repulsion than an ion but more than an apolar molecule and are separated in
order of their polarity (more polar compound eluting earlier). This technique has been used in the analysis of organic acids,
alcohols, glycols, and sugars.
Any suitable detector can be used for the detection and quantitation of analytes by IC. However, two forms of ED, suppressed
conductivity detection (SCD) and pulsed amperometry detection (PAD), have distinct advantages over other detection methods.
They provide the selectivity and sensitivity necessary for the measurement of critical components of fermentation broths without
any pre- or post-column derivatization or complex sample preparation procedures.
When a constant voltage is applied across two electrodes between which the effluent from a column flows, a current is generated
because the effluent contains ions and polar molecules. The current is proportional to the conductivity of the solution, which,
in turn, is proportional to the concentration of ionic species in solution, leading to the detection and quantitation of polar
analytes by a conductivity detector. The problem, however, is that the conductivity of the mobile phase is significantly higher
than the conductivities of the analytes, simply because the concentrations of ions in the mobile phase is 104–105 times higher
than that of the analytes. SCD permits detection and quantitation of analytes by reducing the conductivity of the mobile phase
(baseline conductivity) to nearly zero. It also enhances the conductivity of the analytes, thereby enhancing sensitivity by
about an order of magnitude (2). The peaks of apolar and weakly polar molecules, such as sugars (except the ionic sugars,
e.g., sialic acids, sugar-phosphates), alditols, alcohols, and glycols are not detected or remain close to the noise level,
providing selectivity of detection to the ionic and polar components of the media.
PAD is specific for compounds, such as, carbohydrates, amino acids, alditols, glycols, and alcohols, which can be oxidized
at a selected potential (applied voltage), leaving most of the other compounds undetectable, and thus, providing selectivity
A detailed discussion on the mechanisms of ion-exchange and ion-exclusion chromatographies as well as those of SCD and PAD
is beyond the scope of this article. Interested readers are encouraged to read books and publications that provide details
of the principles and mechanisms of IC, SCD and PAD, including selection of columns and detectors for different types of applications