Manufacturing process for the production of peptides grown in insect cell lines

1 . A method of separating a peptide from an impurity with a molecular weight lower

than said peptide by hydrophobic interaction chromatography, said method comprising:

(a) applying a mixture comprising said peptide and said impurity to a hydrophobic interaction chromatography resin;

(b) eluting said impurity from said resin;

(c) eluting said peptide from said resin; and

(d) collecting an eluate fraction from (c) comprising said peptide, thereby separating said peptide from said impurity.

2 . The method of claim 1 , wherein at least about 50% of said impurity is removed by said method.

3 . The method of claim 2 , wherein at least about 90% of said impurity is removed by said method.

4 . The method of claim 1 , further comprising, prior to step (a):

(e) desalting a mixture comprising said peptide and said impurity, forming a desalted peptide mixture;

(f) eluting said desalted peptide mixture of step (e) from a hydroxyapatite chromatography medium; and

(g) collecting an eluate fraction from (f), comprising said peptide.

5 . The method of claim 1 , wherein said peptide comprises a substantially uniform insect-specific glycosylation pattern.

6 . The method of claim 1 , wherein said peptide is a member selected from erythropoietin, granulocyte colony stimulating factor, GNT1, GalT1, ST3Gal3, CST2, sialidase, GalNAcT2, Core1GalT, ST6GalNAc1, ST3Gal1, and ST3Gal2.

7 . The method of claim 1 , wherein said mixture comprising said peptide is provided by a procedure comprising:

(h) infecting insect cells in an insect cell culture with a recombinant baculovirus comprising a nucleotide sequence encoding said peptide

wherein

(i) said cell culture medium is supplemented with a lipid mixture; and

(ii) said infecting occurs in the culture medium supplemented with said lipid mixture; and

(i) growing the infected insect cells of (h) to produce a culture liquid comprising said peptide encoded by said nucleic acid sequence

wherein said peptide comprises an insect-specific glycosylation pattern.

8 . The method of claim 7 , wherein said lipid mixture is supplemented into said insect cell culture at a percentage of total culture volume equivalent to between about 0.5% to about 3% v/v.

9 . The method of claim 7 , wherein said lipid mixture is added to supplement said insect cell culture from between about 0.5 hours to about 2.0 hours prior to said infecting.

10 . The method of claim 7 , wherein said infecting employs a multiplicity of infection between about 10 −8 to about 1.0.

11 . The method of claim 7 , wherein said lipid mixture comprises: an alcohol, a surfactant, a sterol, a detergent, an anti-oxidant, and a lipid source.

12 . The method of claim 7 , further comprising:

(j) removing cellular debris from said culture liquid to produce a first mixture comprising said peptide;

(k) conditioning said first mixture (j) using a tangential flow filtration cascade;

(l) adjusting pH of conditioned mixture from (k), forming a pH adjusted mixture;

(m) eluting said pH adjusted mixture from (l) from an anion-exchange medium;

(n) collecting an eluate fraction from (m) comprising said peptide;

(o) eluting collected eluate fraction from (n) from a cation-exchange medium;

(p) collecting an eluate fraction from (o) comprising said peptide;

(q) subjecting said eluate fraction from (p) to a low-pH hold procedure, forming a viral-inactivated peptide solution; and

(r) concentrating said viral inactivated peptide solution.

13 . The method of claim 12 , wherein said removing of (j) is accomplished by a member selected from batch centrifugation, continuous centrifugation, filtration, and continuous centrifugation followed by filtration.

14 . The method of claim 13 , wherein said continuous centrifugation is accomplished using a disk-stack centrifuge.

15 . The method of claim 12 , wherein said concentrating of (r) is accomplished by ultrafiltration.

16 . The method of claim 1 , further comprising:

(s) isolating said peptide from (i);

(t) contacting isolated peptide from (s) with a glycosyltransferase and a modified glycosyl donor, comprising a glycosyl moiety which is a substrate for said glycosyltransferase, which is covalently linked to a modifying group, under conditions appropriate for the formation of a covalent bond between said glycosyl moiety of said glycosyl donor and said peptide, thereby producing a modified glycopeptide; and

(u) purifying said modified glycopeptide.

17 . A method of separating a peptide from an impurity by hydroxyapatite chromatography, said method comprising:

(a) desalting a mixture comprising said peptide and said impurity, forming a desalted peptide mixture;

(b) applying said desalted peptide mixture from (a) to a hydroxyapatite resin;

(c) washing said hydroxyapatite resin, removing said impurity from said resin;

(d) eluting said peptide from said resin with an elution buffer; and

(e) collecting an eluate fraction from (d) comprising said peptide, thereby separating said peptide from said impurity.

18 . The method of claim 17 , wherein said desalted mixture has a conductivity between about 0.1 mS/cm and about 4.0 mS/cm.

19 . The method of claim 17 , wherein said elution buffer comprises an amino acid.

20 . The method of claim 19 , wherein said amino acid is glycine.

21 . The method of claim 20 , wherein said glycine is added to said elution buffer at a final concentration of about 5 mM to about 50 mM.

22 . The method of claim 17 , wherein said peptide comprises a substantially uniform insect-specific glycosylation pattern.

23 . The method of claim 17 , wherein said peptide is a member selected from erythropoietin, granulocyte colony stimulating factor, GNT1, GalT1, ST3Gal3, CST2, sialidase, GalNAcT2, Core1GalT, ST6GalNAc1, ST3Gal1, and ST3Gal2.

24 . The method of claim 17 , wherein said mixture comprising said peptide is provided by a procedure comprising:

(f) infecting insect cells in an insect cell culture with a recombinant baculovirus comprising a nucleotide sequence encoding said peptide

wherein

(i) said cell culture is supplemented with a lipid mixture; and

(ii) said infecting occurs in said cell culture supplemented with said lipid mixture; and

(g) growing the infected insect cells of step (f) to produce a culture liquid comprising said peptide encoded by said nucleic acid sequence

wherein said peptide comprises an insect-specific glycosylation pattern.

25 . The method of claim 24 , wherein said lipid mixture is supplemented into said insect cell culture at a percentage of total culture volume equivalent to between about 0.5% to about 3% v/v.

26 . The method of claim 24 , wherein said lipid mixture is added to supplement said insect cell culture from between about 0.5 hours to about 2.0 hours prior to infecting.

27 . The method of claim 24 , wherein said infecting employs a multiplicity of infection between about 10 −8 to about 1.0.

28 . The method of claim 24 , wherein said lipid mixture comprises: an alcohol, a surfactant, a sterol, a detergent, an anti-oxidant, and a lipid source.

29 . The method of claim 24 , further comprising:

(h) removing cellular debris from said culture liquid to produce a first mixture comprising said peptide;

(i) conditioning said first mixture from (h) using a tangential flow filtration cascade;

(j) adjusting pH of conditioned mixture from (i), forming a pH adjusted peptide mixture;

(k) eluting said pH adjusted mixture from 0) from an anion-exchange medium;

(l) collecting an eluate fraction from (k) comprising said peptide;

(m) eluting collected eluate fraction from (1) from a cation-exchange medium;

(n) collecting an eluate fraction from (m) comprising said peptide;

(o) subjecting collected eluate fraction from (n) to a low-pH hold procedure, forming a viral inactivated peptide mixture;

(p) eluting said viral inactivated mixture comprising from a hydrophobic interaction chromatography medium;

(q) collecting an eluate fraction comprising said peptide from (p); and

(r) concentrating said eluate fraction from (q).

30 . The method of claim 29 , wherein said removing of step (h) is accomplished by a member selected from batch centrifugation, continuous centrifugation, filtration, and continuous centrifugation followed by filtration.

31 . The method of claim 30 , wherein said continuous centrifugation is accomplished using a disk-stack centrifuge.

32 . The method of claim 29 , wherein said concentrating of step (r) is accomplished by ultrafiltration.

33 . The method of claim 17 , further comprising:

(s) isolating said peptide from (g);

(t) contacting isolated peptide from (s) with a glycosyltransferase and a modified glycosyl donor, comprising a glycosyl moiety which is a substrate for said glycosyltransferase, which is covalently linked to a modifying group, under conditions appropriate for the formation of a covalent bond between said glycosyl moiety of said glycosyl donor and said peptide, thereby producing a modified glycopeptide; and

(u) purifying said modified glycopeptide.

34 . A method of preparing a viral inactivated peptide mixture by a low-pH hold procedure, said method comprising:

(a) lowering pH of a mixture comprising said peptide;

(b) maintaining said pH of step (a) for a selected period of time; and

(c) raising said pH of said mixture comprising said peptide, forming a viral-inactivated peptide mixture.

35 . The method of claim 34 , wherein said pH of step (a) is lowered to between about pH 2.0 and about pH 4.0.

36 . The method of claim 35 , wherein said pH of step (a) is lowered to between about pH 2.0 and about pH 2.5.

37 . The method of claim 34 , wherein said period of time is selected from between about 30 minutes and about 2 hours.

38 . The method of claim 37 , wherein said period of time is about 1 hour.

39 . The method of claim 34 , wherein said peptide comprises a substantially uniform insect-specific glycosylation pattern.

40 . The method of claim 34 , wherein said peptide is a member selected from erythropoietin, granulocyte colony stimulating factor, GNT1, GalT1, ST3Gal3, CST2, Sialidase, GalNAcT2, Core1GalT, ST6GalNAc1, ST3Gal1, and ST3Gal2.

41 . The method of claim 34 , wherein said mixture comprising said peptide is provided by a procedure comprising:

(d) infecting insect cells in an insect cell culture with a recombinant baculovirus comprising a nucleotide sequence encoding said peptide

wherein

(i) said cell culture is supplemented with a lipid mixture; and

(ii) said infecting occurs in said cell culture supplemented with said lipid mixture; and

(e) growing the infected insect cells of step (d) to produce a culture liquid comprising said peptide encoded by said nucleic acid sequence

wherein said peptide comprises an insect-specific glycosylation pattern.

42 . The method of claim 41 , wherein said lipid mixture is supplemented into said insect cell culture at a percentage of total culture volume equivalent to between about 0.5% to about 3% v/v.

43 . The method of claim 41 , wherein said lipid mixture is added to supplement said insect cell culture from between about 0.5 hours to about 2.0 hours prior to said infecting.

44 . The method of claim 41 , wherein said infecting employs a multiplicity of infection between about 10 −8 to about 1.0.

45 . The method of claim 41 , wherein said lipid mixture comprises: an alcohol, a surfactant, a sterol, a detergent, an anti-oxidant, and a lipid source.

46 . The method of claim 34 , further comprising prior to (a):

(f) removing cellular and other debris from said insect cell culture to produce a first mixture comprising said peptide;

(g) conditioning said first mixture of step (f) using a tangential flow filtration cascade;

(h) adjusting pH of said conditioned mixture of step (g), forming a pH adjusted mixture;

(i) eluting pH adjusted mixture (h) from an anion-exchanger;

(j) collecting an eluate fraction from (i) comprising said peptide;

(k) eluting said eluate fraction from 0) from a cation-exchange medium; and

(l) collecting an eluate fraction from (k) comprising said peptide.

47 . The method of claim 34 , further comprising following (c):

(m) desalting said viral-inactivated peptide mixture of (c), forming a desalted peptide mixture

(n) eluting said desalted peptide mixture from (m) from a hydroxyapatite chromatography medium;

(o) collecting an eluate fraction from (n) comprising said peptide;

(p) subjecting said eluate fraction from (o) to hydrophobic interaction chromatography;

(q) collecting an eluate fraction from (p) comprising said peptide; and

(r) concentrating said eluate fraction from (q) comprising said peptide.

48 . The method of claim 46 , wherein said removing of step (f) is accomplished by a procedure, which is a member selected from batch centrifugation, continuous centrifugation, filtration, and continuous centrifugation followed by filtration.

49 . The method of claim 46 , wherein said continuous centrifugation is accomplished using a disk-stack centrifuge.

50 . The method of claim 47 , wherein said concentrating of step (r) is accomplished by ultrafiltration.

51 . The method of claim 34 , further comprising:

(s) isolating said peptide from (e);

(t) contacting isolated peptide from (s) with a glycosyltransferase and a modified glycosyl donor, comprising a glycosyl moiety which is a substrate for said glycosyltransferase, which is covalently linked to a modifying group, under conditions appropriate for the formation of a covalent bond between said glycosyl moiety of said glycosyl donor and said peptide, thereby producing a modified glycopeptide; and

(u) purifying said modified glycopeptide.

52 . A method of purifying a peptide, said method comprising:

(a) conditioning a mixture comprising said peptide using a tangential flow filtration cascade wherein said conditioning occurs prior to subjecting said mixture to chromatographic purification steps.

53 . The method of claim 52 , wherein said conditioning comprises:

(i) ultrafiltering said mixture across a first ultrafiltration membrane;

(ii) ultrafiltering permeate from step (i) across a second ultrafiltration membrane; and

(iii) collecting retentate from step (ii).

54 . The method of claim 53 , wherein said first ultrafiltration membrane has a molecular weight cutoff of between about 50 kDa and about 150 kDa.

55 . The method of claim 54 , wherein said first ultrafiltration membrane has a molecular weight cutoff of about 100 kDa.

56 . The method of claim 53 , wherein said second ultrafiltration membrane has a molecular weight cutoff of between about 5 kDa and about 15 kDa.

57 . The method of claim 56 , wherein said second ultrafiltration membrane has a molecular weigh cutoff of about 10 kDa.

58 . The method of claim 52 , wherein said peptide comprises a substantially uniform insect-specific glycosylation pattern.

59 . The method of claim 52 , wherein said peptide is a member selected from erythropoietin, granulocyte colony stimulating factor, GNT1, GalT1, ST3Gal3, CST2, sialidase, GalNAcT2, Core1GalT, ST6GalNAc1, ST3Gal1, and ST3Gal2.

60 . The method of claim 52 , wherein said mixture comprising said peptide is provided by a procedure comprising:

(b) infecting insect cells in an insect cell culture with a recombinant baculovirus that comprises a nucleotide sequence encoding said peptide

wherein

(i) said cell culture is supplemented with a lipid mixture; and

(ii) said infecting occurs in the culture supplemented with said lipid mixture; and

(c) growing the infected insect cells of step (a) to produce a culture liquid comprising said peptide encoded by said nucleic acid sequence

wherein

said peptide comprises an insect-specific glycosylation pattern.

61 . The method of claim 60 , wherein said lipid mixture is supplemented into the insect cell culture at a percentage of the total culture volume equivalent to between about 0.5% to about 3% v/v.

62 . The method of claim 60 , wherein said lipid mixture is added to supplement the insect cell culture from between about 0.5 hours to about 2.0 hours prior to said infecting.

63 . The method of claim 60 , wherein said infecting employs a multiplicity of infection between about 10 −8 to about 1.0.

64 . The method of claim 60 , wherein the lipid mixture comprises: an alcohol, a surfactant, a sterol, a detergent, an anti-oxidant, and a lipid source.

65 . The method of claim 60 , further comprising:

(d) removing cellular and other debris from said culture liquid to produce a first mixture comprising said peptide;

(e) adjusting pH of said first mixture comprising said peptide, forming a pH adjusted mixture;

(f) eluting said pH adjusted mixture comprising said peptide from (e) over an anion-exchanger;

(g) collecting an eluate fraction from (f) comprising said peptide;

(h) eluting said eluate fraction from (g) from a cation-exchange medium;

(i) collecting an eluate fraction from (h) comprising said peptide;

(j) subjecting said eluate fraction from (i) to a low-pH hold procedure, forming a viral inactivated peptide mixture;

(k) desalting said viral-inactivated peptide mixture from (j), forming a desalted peptide mixture;

(l) eluting said desalted peptide mixture of (k) from a hydroxyapatite chromatography medium;

(m) collecting an eluate fraction from (l), comprising said peptide;

(n) subjecting said eluate fraction from (m) to hydrophobic interaction 1 9 chromatography;

(o) collecting an eluate fraction from (n), comprising said peptide; and

(p) concentrating said eluate fraction.

66 . The method of claim 65 , wherein said removing of step (d) is accomplished by a member selected from batch centrifugation, continuous centrifugation, filtration, and continuous centrifugation followed by filtration.

67 . The method of claim 66 , wherein said continuous centrifugation is accomplished using a disk-stack centrifuge.

68 . The method of claim 65 , wherein said concentrating of step (p) is accomplished 2 by ultrafiltration.

69 . The method of claim 52 , further comprising:

(q) isolating said peptide from (c);

(r) contacting isolated peptide from (q) with a glycosyltransferase and a modified glycosyl donor, comprising a glycosyl moiety, which is a substrate for said glycosyltransferase, which is covalently linked to a modifying group, under conditions appropriate for the formation of a covalent bond between said glycosyl moiety of said glycosyl donor and said peptide, thereby producing a modified glycopeptide; and

(s) purifying said modified glycopeptide.

70 . A method of purifying a peptide, said method comprising:

(a) removing cellular and other debris from a cell culture comprising said peptide, to produce a first mixture comprising said peptide;

(b) conditioning said first mixture of step (a) using a tangential flow filtration cascade, forming a conditioned mixture;

(c) adjusting pH of said conditioned mixture of step (b), forming a pH adjusted mixture;

(d) eluting said pH-adjusted conditioned mixture from step (c) from an an anion-exchange medium;

(e) collecting an eluate fraction from (d) comprising said peptide;

(f) eluting said eluate fraction from (e) from a cation exchange medium;

(g) collecting an eluate fraction from (f) comprising said peptide;

(h) subjecting said eluate fraction of (g) to a low-pH hold procedure producing a viral inactivated mixture comprising said peptide;

(i) desalting said viral inactivated mixture of step (h), forming a desalted mixture;

(j) eluting said desalted mixture of step (i) from a hydroxyapatite chromatography medium;

(k) collecting an eluate fraction comprising said peptide from (j);

(l) subjecting the eluate fractions of step (k) to hydrophobic interaction chromatography;

(m) collecting an eluate fraction from (l) comprising said peptide; and

(n) concentrating said eluate fraction from (m).

71 . A lipid composition for use in conjunction with a baculovirus expression vector system, the composition comprising: an alcohol, a surfactant, a sterol, a detergent, an anti-oxidant, and a lipid source.

72 . The composition of claim 71 , comprising:

said alcohol in an amount between about 5% v/v to about 20% v/v;

said surfactant in an amount between about 5% w/v and about 15% w/v;

said sterol in an amount between about 0.02% to about 0.06% w/v;

said detergent in an amount between about 0.1% w/v to about 0.3% w/v,;

said anti-oxidant in an amount between about 0.01% w/v to about 0.05% w/v; and

said lipid source in an amount between about 0.05% w/v to about 0.25% w/v.