Size enhanced hemoglobins: surface decoration and crosslinking of the protein with polyoxy alkylene glycols

A hemoglobin composition comprising hemoglobin (Hb) decorated with one or more PEG molecules, wherein the Hb-PEG has the formula

• Hb-(CO—NH—CHR—CO—W—CH2—CH2—[O—CH2—CH2]n—R′)m
• wherein n is an integer from about 125 to about 500, m is an integer from 1 to 10, W is NH or O, R is an amino acid side chain, R′ is selected from the group consisting of OH, OCH3, CH2OH, CH2OCH3, CH2CH2OH, and CH2CH2OCH3, and wherein at least one PEG is bonded to the Hb at Glu-43(β).

A hemoglobin composition comprising at least one hemoglobin molecule (Hb), crosslinked by one or more PEG molecules, wherein the crosslinked Hb has the formula

• Hb-CO—NH—CHR—CO—W—CH2—CH2- [O—CH2—CH2]n—NH—CO—W′—CHR—NH—CO—Hb′
• wherein Hb and Hb′ are the same or different hemoglobin molecule, n is an integer from about 15 to about 250, W and W′ are each independently NH or O, R is an amino acid side chain, and HbA and/or HbA′ is bonded to the PEG with an amide linkage at Glu-43(β).

A method of producing a hemoglobin comprising a polyalkylene glycol, wherein the polyalkylene glycol is polypropylene glycol or polyethylene glycol (PEG), the method comprising mixing in a suitable buffer (a) the hemoglobin, (b) a carbodiimide, and (c) a nucleophilic polyalkylene glycol with a terminal amine having a pKa below 9, and incubating the mixture under conditions and for a time sufficient for the polyalkylene glycol to covalently bond to the hemoglobin at Glu-43(β).

A method of producing a hemoglobin comprising a polyethylene glycol (PEG), the method comprising mixing in a suitable buffer (a) the hemoglobin, (b) a carbodiimide, and (c) a nucleophilic PEG with a terminal amine having a pKa below 9, and incubating the mixture under conditions and for a time sufficient for the PEG to covalently bond to the hemoglobin at Glu-43(β), wherein the nucleophilic PEG has the formula

• H2N—CHR—CO—W—CH2—CH2—[O—CH2—CH2]n—R′
• wherein n is an integer from about 125 to about 500, W is NH or O, R is an amino acid side chain, and R′ is selected from the group consisting of OH, OCH3, CH2OH, CH2OCH3, CH2CH2OH, and CH2CH2OCH3.

A hemoglobin comprising a polyalkylene glycol, wherein the polyalkylene glycol is polypropylene glycol or polyethylene glycol (PEG), and wherein the polyalkylene glycol is covalently bonded to the hemoglobin at the α-amino of a Val-1(β).

A hemoglobin composition comprising hemoglobin A (HbA) decorated with one or more PEG molecules, wherein the HbA-PEG has the formula

• HbA-(NH—CH—[CH2]p[NH]q—CH2—CH2—[O—CH2—CH2]n—R)m
• wherein p is an integer from 2 to 6, q is 1 or 2, n is an integer from about 125 to about 500, m is an integer from 1 to 10, and R is selected from the group consisting of OH, OCH3, CH2OH, CH2OCH3, CH2CH2OH, and CH2CH2OCH3.

A hemoglobin composition comprising a hemoglobin (Hb) crosslinked by one or more PEG molecules, wherein the crosslinked Hb has the formula

• Hb-NH—CH—[CH2]p—[NH]q—CH2—CH2 [O—CH2—CH2]p—[NH]q—[CH2]p—CH—NH-Hb′
• wherein Hb and Hb′ are the same or different hemoglobin molecule, each p is independently an integer from 2 to 6, each q is 1 or 2, and n is an integer from about 15 to about 250.

A method of producing a hemoglobin comprising a polyalkylene glycol, wherein the polyalkylene glycol is a polypropylene glycol or a polyethylene glycol (PEG), and wherein the PEG is covalently bonded to the hemoglobin at the α-amino of a Val-1(β), the method comprising incubating the hemoglobin with a polyalkylene glycol aldehyde and a borohydride under conditions and for a time sufficient for the polyalkylene glycol aldehyde to bond to the hemoglobin at the Val-1(β).

A method of producing a hemoglobin comprising a polyethylene glycol (PEG), wherein the PEG is covalently bonded to the hemoglobin at the α-amino of a Val-1(β), the method comprising incubating the hemoglobin with a PEG aldehyde and a borohydride under conditions and for a time sufficient for the PEG to bond to the hemoglobin at the Val-1(β), and wherein the PEG aldehyde has the formula

• HOC-[CH2]p—[NH]q—CH2—CH2—[O—CH2—CH2]n—R
• wherein p is an integer from 2 to 6, q is 1 or 2, n is an integer from about 125 to about 500, and R is selected from the group consisting of OH, OCH3, CH2OH, CH2OCH3, CH2CH2OH, and CH2CH2OCH3.

A method of producing a hemoglobin comprising a polyethylene glycol (PEG), wherein the PEG is covalently bonded to the hemoglobin at the α-amino of a Val-1(β) and crosslinks the hemoglobin intramolecularly or intermolecularly, the method comprising incubating the hemoglobin with a PEG aldehyde and a borohydride under conditions and for a time sufficient for the PEG to bond to the hemoglobin at the Val-1(β), wherein the PEG aldehyde has the formula

• OHC—[CH2]p—[NH]q—CH2—CH2—[O—CH2—CH2]n—[NH]q—[CH2]p—CHO
• wherein each p is independently an integer from 2 to 6, each q is independently 1 or 2, and n is an integer from about 15 to about 250.

A hemoglobin comprising a polyalkylene glycol, wherein the polyalkylene glycol is a polypropylene glycol or a polyethylene glycol (PEG), wherein the polyalkylene glycol is covalently bonded to the hemoglobin at a thiol moiety through a phenylsuccinimido linkage, and wherein no polyalkylene glycol is covalently bonded to a Cys-93(β).

A hemoglobin comprising a polyethylene glycol (PEG), wherein the PEG is covalently bonded to the hemoglobin at a thiol moiety through a phenylsuccinimido linkage, and wherein no PEG is covalently bonded to a Cys-93(β), the hemoglobin having the formula

• Hb-S—Y—R—CH2—CH2—[O—CH2—CH2]n—R′—Y′—S-Hb′
• wherein n is an integer from about 15 to about 250, R and R′ are the same or different and are carbamate, urea, or amide, Y and Y′ are the same or different and are 4-phenylsuccinimido or 3-phenylsuccinimido, and Hb and Hb′ are the same or different hemoglobin molecule.

An improved PEGalated hemoglobin comprising a PEG bonded to a thiol group of the hemoglobin through a phenylsuccinimido linkage, wherein the improvement comprises an absence of a PEG bonded to a Cys-93(β).

In a method for producing a PEGalated hemoglobin comprising PEG bonded to a thiol moiety of the hemoglobin through a phenylsuccinimido linkage, the method comprising treating the hemoglobin with a maleidophenyl PEG and iminothiolane under conditions and for a time sufficient for the PEG to conjugate to the thiol moiety of the hemoglobin through a phenylsuccinimido linkage, the improvement comprising conjugating a reagent to a Cys-93(β) sulfhydryl group of the hemoglobin by a reversible method before treating the hemoglobin with the PEG, then removing the reagent after treating the hemoglobin with the PEG.