Immobilized cobalt affinity chromatography provides a novel, efficient method for herpes simplex virus type 1 gene vector purification.
Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early-phase clinical trials, emphasizing the need for high-yield, scalable manufacturing processes that result in virus preparations that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu2+, Zn2+, Ni2+, and Co2+) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA.
Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Canping
C
Jiang
James B
JB
Wechuck
William F
WF
Goins
David M
DM
Krisky
Darren
D
Wolfe
Mohammad M
MM
Ataai
Joseph C
JC
Glorioso
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
D002413:Cations, Divalent; D011506:Proteins; D011993:Recombinant Fusion Proteins; D003035:Cobalt; D004247:DNA