Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn2+ and -Co2+ Affinity Chromatography

Verinaud, Claudia Iwashita; Feliciano, Gabriel Pinna; Carvalho, Roberta Rodrigues de; Lopes, Alexandre P. Y.; Raw, Isaı́as; Martins, Elizabeth A. L.; Cheng, Elisabeth

doi:10.1007/s10337-017-3345-8

Cited by 2 publications

(7 citation statements)

References 19 publications

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“…Protein C, on the other hand, was recovered with a lower yield (44% and 56%) and purification factor (126 and 162). These results are in accordance with the result obtained with the elution of this protein by 500 mM NaCl [ 16 ]. In contrast, recombinant Protein C could be recovered with high yield (85%) (and 96% purity) from culture media on Q Sepharose FF using the Ca 2+ gradient [ 23 ].…”

Section: Discussionsupporting

confidence: 92%

“…The results obtained in this work are in line with this result. On the other hand, the purification factors obtained in this work, regardless the resin used, were much higher than those described in the work of Verinaud et al [ 16 ] (110 ± 11). These results indicate that the CaCl 2 gradient step did not have a negative affect on FVIII activity and significantly improved the purification factor.…”

Section: Discussioncontrasting

confidence: 75%

“…The yield and the purification factor of FVIII in each experiment are shown in Table 1 . In the purification of plasma on ANX Sepharose FF without the CaCl 2 gradient step, the FVIII recovered activity in fraction “500” described by Verinaud et al [ 16 ] was 69 ± 18. The results obtained in this work are in line with this result.…”

Section: Discussionmentioning

confidence: 99%

“…Anion exchange resins are widely used in the plasma fractionation processes [ 3 , 12 , 13 , 14 , 15 ] due to their high capacity, good yields and low cost compared to other chromatographic techniques. Upon loading whole plasma directly onto an anion exchange column, without the cryoprecipitation step, FVIII and PCC are strongly adsorbed and cannot be eluted separately using NaCl gradient [ 16 , 17 ]. A second column was needed to separate FVIII from PCC proteins, and it was successfully achieved by immobilized metal affinity chromatography [ 16 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…Upon loading whole plasma directly onto an anion exchange column, without the cryoprecipitation step, FVIII and PCC are strongly adsorbed and cannot be eluted separately using NaCl gradient [ 16 , 17 ]. A second column was needed to separate FVIII from PCC proteins, and it was successfully achieved by immobilized metal affinity chromatography [ 16 , 18 ]. Conventional affinity chromatography involves the immobilization of a ligand for which the protein of interest has an affinity.…”

Section: Introductionmentioning

confidence: 99%

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Non-Cryoprecipitation Separation of Coagulation FVIII and Prothrombin Complex Proteins by Pseudoaffinity Calcium Elution Chromatography Using Anion Exchange Resin

Feliciano

Arimori²,

Nakao³

et al. 2022

Pharmaceuticals

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Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

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Section: Discussionsupporting

confidence: 92%

Section: Discussioncontrasting

confidence: 75%

Section: Discussionmentioning

confidence: 99%