TY - JOUR
T1 - Mono-PEGylated lysozyme purification with increased productivity and isomer differentiation through heparin monolith chromatography
AU - Mejía-Manzano, Luis Alberto
AU - Campos-García, Víctor R.
AU - Perdomo-Abúndez, Francisco C.
AU - Medina-Rivero, Emilio
AU - González-Valdez, José
N1 - Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/7/15
Y1 - 2022/7/15
N2 - PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.
AB - PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.
KW - Affinity monolith chromatography
KW - Heparin
KW - Lysozyme PEGylation reaction
KW - Mono-PEGylated lysozyme
KW - Reductive alkylation
UR - http://www.scopus.com/inward/record.url?scp=85132455511&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2022.123323
DO - 10.1016/j.jchromb.2022.123323
M3 - Artículo
C2 - 35700648
AN - SCOPUS:85132455511
SN - 1570-0232
VL - 1204
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
M1 - 123323
ER -