Separation of protein charge variants by ultrafiltration

Mareia Frost Ebersold, Andrew Zydney

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine ε-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.

Original languageEnglish (US)
Pages (from-to)543-549
Number of pages7
JournalBiotechnology Progress
Volume20
Issue number2
DOIs
StatePublished - Mar 2004

Fingerprint

myoglobin
Ultrafiltration
ultrafiltration
Myoglobin
biopharmaceuticals
Membranes
electrostatic interactions
anhydrides
permeates
sieving
succinic acid
lysine
Proteins
proteins
Static Electricity
Recombinant Proteins
Lysine
amino acids
Amino Acids
Therapeutics

All Science Journal Classification (ASJC) codes

  • Food Science
  • Biotechnology
  • Microbiology

Cite this

Ebersold, Mareia Frost ; Zydney, Andrew. / Separation of protein charge variants by ultrafiltration. In: Biotechnology Progress. 2004 ; Vol. 20, No. 2. pp. 543-549.
@article{3efd904ed2a24d3b837e9feeb9afe314,
title = "Separation of protein charge variants by ultrafiltration",
abstract = "The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine ε-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90{\%} yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.",
author = "Ebersold, {Mareia Frost} and Andrew Zydney",
year = "2004",
month = "3",
doi = "10.1021/bp034264b",
language = "English (US)",
volume = "20",
pages = "543--549",
journal = "Biotechnology Progress",
issn = "8756-7938",
publisher = "John Wiley and Sons Ltd",
number = "2",

}

Separation of protein charge variants by ultrafiltration. / Ebersold, Mareia Frost; Zydney, Andrew.

In: Biotechnology Progress, Vol. 20, No. 2, 03.2004, p. 543-549.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Separation of protein charge variants by ultrafiltration

AU - Ebersold, Mareia Frost

AU - Zydney, Andrew

PY - 2004/3

Y1 - 2004/3

N2 - The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine ε-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.

AB - The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine ε-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.

UR - http://www.scopus.com/inward/record.url?scp=1842713256&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842713256&partnerID=8YFLogxK

U2 - 10.1021/bp034264b

DO - 10.1021/bp034264b

M3 - Article

VL - 20

SP - 543

EP - 549

JO - Biotechnology Progress

JF - Biotechnology Progress

SN - 8756-7938

IS - 2

ER -