Naslov
Anion-exchange chromatography in the batch mode as a method for purificaion of equine IgGs from the plasma

Autori
Tunjić, Monika ; Brgles, Marija ; Kurtović, Tihana ; Cajner, Hrvoje ; Halassy, Beata

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
2017 Annual Meeting of the Croatian Immunological Society with EFIS on Tour / - , 2017, 67-67

Skup
2017 Annual Meeting of the Croatian Immunological Society with EFIS on Tour

Mjesto i datum
Zagreb, Hrvatska, 20.10.2017. - 21.10.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Podatak o recenziji nije dostupan

Ključne riječi
Equine immunoglobulins ; Anion-exchange chromatography ; Design of Experiments ; Hyperimmune plasma

Sažetak
Background: Immunoglobulin–derived biopharmaceutical products (like antivenoms) should not contain polymer molecules. An appearance of polymers during storage period indicates product instability. It has been shown that purification steps involving precipitation of IgGs during downstream processing of plasma might disturb the tertiary structure of immunoglobulins, making them more prone to polymerization. That is the reason why we investigated the possibilites of purifying IgGs by protocols that render them in solution, avoiding their precipitation and temporary binding to matrices of any kind. Aim: In a hyperimmune horse plasma (HP), IgGs have higher pI values than most of other plasma proteins, particularly albumin, the most abundant non-IgG protein. Thus, methods of separation on the basis of pI differences have been considered. Driven by the idea to keep IgGs constantly in solution during purification, we investigated the possibility of purifying them by anion- exchange chromatography in the batch mode. Materials and methods: Toyopearl SuperQ-650M was used as anionic exchanger, and its binding capacity for pure albumin and IgG precisely determined under anticipated experimental conditions for IgG purification from HP. Depletion of albumin from HP was tested by alternating between the following conditions: pH (7 and 8), buffer ionic strength (35 mM and 120 mM buffer), dilution of plasma (5x and 20x) and the quantity of ion exchanger used (volumes of ion exchanger were set at 30% and 60% of the total resin albumin binding capacity). Total protein concentrations were measured using Ehresmann's method and IgG concentrations were determined using ELISA. One-dimensional SDS- PAGE and SEC-HPLC were used to determine the purity of differently treated plasma fractions. Results: The best purification efficiency was achieved in buffers at pH 8, containing 35 mM NaCl, with IgG purity of 81.2%. However, yields did not exceed 50%. Inadequate separation of IgGs from albumin was connected to an abundance of polymers as determined by SEC-HPLC in the samples where higher NaCl concentration was used. The difference in dilution and ion exchanger quantity had no apparent effect on the separation of IgG and albumin. Conclusions: This mode of ion-exchange chromatography can be used to purify IgG up to 80% purity under some experimental conditions, but with huge losses (up to 50%). Such results on purity and yield are inferior to other known purification procedures, so further investigation and optimization is to be done.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

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