4. Discussion

The availability of a quantitation method for human plasma, Cohn fractions, and Anti-D gammaglobulin has a relevant importance for the process control of the Anti-D IgG production. Pharmaceutical companies are employing sensitive EIA, RIA, and flow cytometry techniques [10, 11, 15, 20, 24] for the quantitation of Anti-D level in finished products, the use in Anti-D gammaglobulin manufacturing process control have no be reported.

Highly significant correlations were observed between the flow cytometry test Anti-D values and EIA values determined in different matrices (Serum, Fraction II,

Flow Cytometry Assay for Quantitation of Therapeutical Anti-D IgG during Process Control…

For the case of process samples (Fraction II, semi-processed), strong correlations were observed between the flow cytometry test and the EIA values. A potential advantage of the flow cytometry assay could be the higher sensitivity presented

It is concluded that the flow cytometry method has advantages over the EIA and

1 Blood Products Laboratory National University of Cordoba, Córdoba, Argentina

2 Group Quality and Regulatory Affairs, School of Chemistry, Catholic University

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: sergio.oviedo@unc.edu.ar

provided the original work is properly cited.

RIA method as a substitute for the present standard method.

Semi-elaborated, gamma globulin).

DOI: http://dx.doi.org/10.5772/intechopen.89614

than the EIA assay.

Author details

Sergio A. Oviedo1,2

of Cordoba, Argentina

63

A rapid and low-cost flow cytometry test applied in the control of the industrial process of large-scale production obtaining Anti-D gammaglobulin has been described. This responded to the need of an alternative method to EIA and RIA in the quantitation of Anti-D in different process samples (human plasma, Chon fractions, gammaglobulin) [29].

According to this, the threshold of the assay was adjusted to select the level of antibody content, which will be accepted, to produce an immunoglobulin with a potency of 250 μg/vial (1250 IU/vial), according to the regulatory requirements [13, 14, 28]. To validate the assay, chemical and physical-chemical factors that affect the binding of antigen-antibody influence in the red blood cells were determined. The spatial conditions of cell packing did not affect the assay. The adsorption of antibodies to the cell surface was 1000 times greater when the incubation temperature was 37°C without this affecting the dissociation rate [9]. The different fractions behave similarly in the assay conditions, and there was no interference by autoagglutination at the concentration of red blood cells used (Figure 7). The analytical quality assurance of the trial showed that the method presents good reproducibility, repeatability (1–7.5 VC%), and correlation with the standard curve (r: 0.9267). The assay is specific and recovery is 95% of the known Anti-D concentration values. For validation our strategy was based on an international guide accepted by manufacturers and control laboratories [13, 30]. The validation method meets the requirements according to the good manufacturing practices in the pharmaceutical industry. A validation methodology was followed to meet the special requirements of standardization of the good manufacturing practices in the pharmaceutical industry [30, 31].

Biological variability, red blood cells, and matrices were considered in a validation protocol according to international guidelines [14, 30].
