Influencia de los parámetros de purificación por cromatografía de afinidad en Proteína A en los atributos críticos de calidad del anticuerpo monoclonal anti - CD20
Archivos
Fecha
2019-06
Autores
Pargas Ferrer, Elizabeth
Título de la revista
ISSN de la revista
Título del volumen
Editor
Universidad Central "Marta Abreu " de Las Villas
Resumen
El anticuerpo monoclonal anti
-
CD20 se utiliza para tratar enfermedades oncogénicas y
autoinmunes con proliferación patológica de linfocitos B. El Centro de I
nmunología
Molecular obtuvo el
biosimilar
anti
-
CD20 1B8 (CimaBior®), pero se produjeron cambios
aminoacídicos respecto al producto de referencia, esto lo
invalidó para su
comercialización. Por tanto, se desarrolló un nuevo biosimilar con el error corregido
.
Como parte del nuevo desarrollo, se determinó
la influencia del pH y la molaridad de las
soluciones de equilibrio y elución de la purificación por cromatografía de afinidad en
Proteína A: a escala micro analítica en el recobrado y a escala de laboratorio
en la las
propiedades físico
-
químicas como heterogeneidad de talla y cargas, estructura secundaria
y terciaria. Se varió el pH y la conductividad de acuerdo a un diseño factorial para la
purificación a escala micro analítica y a escala de laboratorio. Las
propiedades físico
-
químicas se evaluaron mediante técnicas analíticas. Resultó que
los rangos de estos
parámetros que garantizan los atributos críticos de calidad del nuevo biosimilar fueron:
pH
6,8
–
7,
4 y molaridad 150 mM de la solución de equilibrio, y p
H 3,25 para la de elución. A
escala micro analítica, solo el pH de la solución de elución influyó significativamente en el
recobrado. A escala de laboratorio, se determinó q
ue a pH de elución inferior a 3,
0 se
formaron agregados de alto peso molecular y productos de degradación. El pH de la
solución de elución influyó significativamente en el porciento de isoformas ácidas y en la
estructura secundaria y terciaria del anticuerpo.
Monoclonal antibody anti - CD20 is used to treat B - cell malignancies and B - cell mediated autoimmune diseases. The Center of Molecular Immunology obtained the biosimilar anti - CD20 1B8 (CimaBior®), but there were amino acid changes with respect to refer ence product; invalidating 1B8 (CimaBior®) for its marketing. Therefore, a new biosimilar was developed with the error corrected. As part of the process, the influence of pH and molarity of equilibrium and elution solutions of the purification by Protein A affinity chromatography, were determined. The influence on recovery was determined at the micro - analytical scale; whereas the influence on physico - chemical properties such as height and charge heterogeneity, secondary and tertiary structure, were determin ed at the laboratory scale. The pH and conductivity were varied according to a factorial design for purification at micro - analytical and laboratory scales. The physico - chemical properties were evaluated by analytical techniques. The ranges of these paramet ers, that guarantee the critical quality attributes of the new biosimilar, were: pH 6,8 - 7, 4 and molarity 150 mM for the equilibrium solution, and pH 3, 25 for the elution solution. At micro - analytical scale, only the pH of elution influenced significantly i n the recovery. At laboratory scale, high molecular weight aggregates and degradation products were formed with the pH of elution lower than 3,0. The pH of elution influenced significantly in the percentage of acid isoforms and the antibody secondary and t ertiary structures.
Monoclonal antibody anti - CD20 is used to treat B - cell malignancies and B - cell mediated autoimmune diseases. The Center of Molecular Immunology obtained the biosimilar anti - CD20 1B8 (CimaBior®), but there were amino acid changes with respect to refer ence product; invalidating 1B8 (CimaBior®) for its marketing. Therefore, a new biosimilar was developed with the error corrected. As part of the process, the influence of pH and molarity of equilibrium and elution solutions of the purification by Protein A affinity chromatography, were determined. The influence on recovery was determined at the micro - analytical scale; whereas the influence on physico - chemical properties such as height and charge heterogeneity, secondary and tertiary structure, were determin ed at the laboratory scale. The pH and conductivity were varied according to a factorial design for purification at micro - analytical and laboratory scales. The physico - chemical properties were evaluated by analytical techniques. The ranges of these paramet ers, that guarantee the critical quality attributes of the new biosimilar, were: pH 6,8 - 7, 4 and molarity 150 mM for the equilibrium solution, and pH 3, 25 for the elution solution. At micro - analytical scale, only the pH of elution influenced significantly i n the recovery. At laboratory scale, high molecular weight aggregates and degradation products were formed with the pH of elution lower than 3,0. The pH of elution influenced significantly in the percentage of acid isoforms and the antibody secondary and t ertiary structures.
Descripción
Palabras clave
Anticuerpo Monoclonal, Biosimilar, Rituximab, CimaBior®, Monoclonal Antibody