Screening and characterization of biopharmaceuticals by high‐performance capillary electrophoresis with laserinduced native fluorescence detection

Abstract

High-performance capillary electrophoresis (HPCE) with laser-induced native fluorescence (LIF) detection is used to address significant problems in the quality control of biopharmaceuticals. All of the biopharmaceuticals studied can be detected at subnanomolar levels with linear dynamic ranges of at least 3 orders of magnitude. HPCE/LIF can determine impurities in "purified" biopharmaceuticals present in amounts less than 0.01% (i.e., at 4 x 10(-11) M) that of the major component. With HPCE/LIF, detection sensitivity is thus no longer a concern in the assaying of active ingredients in biopharmaceutical dosage formulations. The peptide mapping of biopharmaceuticals present at 1 x 10(-7) M (or an injected limit of detection of 60 amol) is presented. Also, kinetic information on the reaction of a recombinant enzyme-drug with its substrate present at the micromolar level has been extracted from electropherograms acquired in real-time.

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

“…The presence of impurities in purified human factor XIII and in monoclonal antibody against human IL‐2 receptor was demonstrated by Lee et al. . It was shown that the UVLIF electropherogram was much simpler than the one obtained with a UV absorbance detector due to the selectivity of fluorescence.…”

Optimization of CZE for analysis of phytochemical bioactive compounds

“…Therefore, the development of techniques that can improve detection has been a principal area of CE research. Up to date, electrochemical (EC) [62,63,67,85,132,[161][162][163][164][165][166][167][168], LIF [22,29,31,169], chemiluminescence (CL) [170][171][172][173][174][175], electrochemiluminescence (ECL) [176,177] and MS [11,89,145,[178][179][180][181][182][183][184][185][186] detections have been successfully applied for CZE analysis.…”

“…It has been widely applied for analysis of pharmaceuticals, including relatively small synthetic drugs [16][17][18][19], DNA analogs (antisense DNA drugs) [20][21][22], naturally produced drugs (traditional medicines) [1,[23][24][25][26][27][28][29][30], and biopharmaceuticals (peptides, proteins, etc.) [31][32][33]. Among various modes of CE such as CZE, MEKC, CGE, CIEF, capillary isotachophoresis (CITP), CEC and non-aqueous CE, CZE is the most frequently used method because it is the simplest and most versatile CE mode (Fig.…”

Capillary electrophoresis with laser-induced fluorescence detection for natively fluorescent analytes

“…Examples of proteins analysed in single cells are insulin [12] and several haemoglobin variants in single erythrocytes by CZE [13,14], or capillary isoelectric focusing (cIEF) [15]. Furthermore, various biopharmaceuticals [16], catecholamines [17], and nucleic acids [18] were analysed by CE with UV-LIF detection, and -last but not least -it can also be applied to a variety of organic compounds of environmental interest, as will be shown below. Another interesting line of development concerns intracavity frequency doubling of the visible lines of the Ar + laser, producing for instance CW output at 257 nm.…”