Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis.

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.

Comparison of alpha-1-acid glycoprotein isoforms from healthy and cancer patients by capillary IEF.

alpha-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same individual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein.

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.

Immunochromatographic removal of albumin in erythropoietin biopharmaceutical formulations for its analysis by capillary electrophoresis.

Human serum albumin (HSA) is added to some pharmaceutical preparations as an excipient. This is the case for some of the commercial preparations of recombinant erythropoietin (rEPO). Differences in the number of the sialic acid moieties in the different rEPO glycoforms confer to these forms different net charges and different bioactivity. Knowledge of the isoforms present in each pharmaceutical product is then of interest. Differences in net charge of the rEPO forms make possible their separation by electrophoretical methods. However it has been observed in our laboratory that the amount of HSA usually present in these drug formulations interferes or even precludes separation of rEPO bands by capillary zone electrophoresis (CZE). In this work, an immunochromatographic method to remove HSA from rEPO biopharmaceutical formulations and a procedure to concentrate the sample that is needed to be performed prior to the analysis by CZE are developed. A home-made computer program to compare the percentage of correct assignments of electrophoretical bands provided by different migration parameters is used to study the effect of HSA remaining in samples on the accuracy of assignment of rEPO bands. When there exists a residual concentration of HSA in the sample (<2mg/ml) only the effective electrophoretic mobility is a reliable migration parameter to assign rEPO bands with a 100% of correct assignment. This parameter allows the correct assignment of bands of rEPO from pharmaceutical products formulated with HSA after immunochromatographic removal of HSA. Electrophoretical bands found in epoetin alpha, one of the commercial formulations of rEPO, are independent of the molecular mass of the excipients. The methodology used in this work for the analysis by CZE and the assignment of rEPO isoforms, as well as for the immunochromatographic HSA removal in the pharmaceutical products could be of high interest for the health authorities to control the quality of the product in marketing surveillance studies and for the quality control laboratories of the manufacturers.

CZE of human alpha-1-acid glycoprotein for qualitative and quantitative comparison of samples from different pathological conditions.

Alpha-1-acid glycoprotein (AGP) presents different forms, which may arise from differences in the amino acid sequence and/or in the glycosidic part of the protein. Changes in forms of AGP have been described in literature as a possible tumor marker. While most previous works have approached the study of glycopeptides and/or glycans obtained after fragmentation of the protein, in this work, a CZE method is developed to separate up to eleven peaks of intact forms of AGP. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Electropherograms of AGP samples purified from sera of cancer patients and healthy donors are qualitatively and quantitatively compared. Percentages of correct assignment of AGP peaks close to 100% are achieved by using either the migration time of each peak relative to that of the EOF marker or the effective electrophoretic mobility of the peaks. The computer program permits to select, among different hypotheses for peak allotment, that one providing the highest accuracy of assignment. In this way, some peaks with different charge-to-mass ratio and a different distribution of area percentage of AGP forms are observed when comparing samples from sick and healthy individuals. Thus, a method that permits to compare AGP forms existing in sera of individuals with different pathophysiological situations has been developed. A potential for using AGP forms analyzed by CZE as a disease marker and for using this technique for screening purposes is envisaged.

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis.

Capillary zone electrophoresis of samples of recombinant human erythropoietin is performed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of alpha- and beta-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin alpha, the novel erythropoiesis-stimulating protein.