U.S. patent application number 11/264345 was filed with the patent office on 2006-05-18 for regeneration of chromatographic stationary phases.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Per Larsen, Eigil Schroder Rasmussen, Ole Schou.
Application Number | 20060102561 11/264345 |
Document ID | / |
Family ID | 36118397 |
Filed Date | 2006-05-18 |
United States Patent
Application |
20060102561 |
Kind Code |
A1 |
Larsen; Per ; et
al. |
May 18, 2006 |
Regeneration of chromatographic stationary phases
Abstract
Process for regenerating a chromatographic stationary phase
Inventors: |
Larsen; Per; (Holte, DK)
; Schou; Ole; (Stensved, DK) ; Rasmussen; Eigil
Schroder; (Kobenhavn K, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;PATENT DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
36118397 |
Appl. No.: |
11/264345 |
Filed: |
September 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/DK04/00234 |
Apr 2, 2004 |
|
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11264345 |
Sep 30, 2005 |
|
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Current U.S.
Class: |
210/656 ;
210/198.2; 210/502.1 |
Current CPC
Class: |
B01D 15/203 20130101;
B01J 20/285 20130101; B01D 15/20 20130101; A61P 3/10 20180101; B01J
20/3425 20130101; B01J 20/3475 20130101; B01J 20/283 20130101; C07K
1/20 20130101; B01J 20/3433 20130101; B01J 20/34 20130101 |
Class at
Publication: |
210/656 ;
210/198.2; 210/502.1 |
International
Class: |
B01D 15/08 20060101
B01D015/08 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 2003 |
DK |
PA 2003 00536 |
Jan 26, 2004 |
DK |
PA 2004 00098 |
Claims
1. A process for regenerating a chromatographic stationary phase
wherein said chromatographic stationary phase is contacted with a
regeneration solution comprising at least one organic acid and less
than about 75% w/w water.
2. The process according to claim 1, wherein the regeneration
solution comprises at least one organic acid and less than about 1%
w/w water.
3. The process according to claim 1, wherein the concentration of
said organic acid is at least about 25% w/w.
4. The process according to claim 1, wherein said organic acid is
formic acid.
5. The process according to claim 1, wherein said organic acid is
acetic acid.
6. The process according to claim 1, wherein said regeneration
solution comprises an organic solvent.
7. The process according to claim 6, wherein said organic solvent
is ethanol.
8. The process according to claim 6, wherein said organic solvent
is 2-propanol.
9. The process according to claim 6, wherein said organic solvent
is acetonitrile.
10. The process according to claim 6, wherein said organic solvent
is selected from the group consisting of methanol, 1-propanol, and
hexylene glycol.
11. The process according to claim 1, wherein said regeneration
solution contains less than 0.5% water.
13. The process according to claim 1, wherein said chromatographic
stationary phase is contacted with said regeneration solution
inside the chromatographic column.
14. The process according to claim 13, wherein said chromatographic
stationary phase is regenerated without repacking the column.
15. The process according to claim 13, wherein said chromatographic
stationary phase is fluidized during said regeneration.
16. The process according to claim 13, wherein the chromatographic
eluent or equilibrium buffer is displaced by a water miscible
organic solvent before said chromatographic stationary phase is
contacted with said regeneration solution.
17. The process according to claim 16, wherein said organic solvent
is also present in the chromatographic eluent or equilibrium
buffer.
18. The process according to claim 16, wherein said water miscible
organic solvent is also present in the regeneration solution.
19. The process according to claim 1, wherein said chromatographic
stationary phase is contacted with said regeneration solution
outside the chromatographic column.
20. The process according to claim 1, wherein said chromatographic
stationary phase is a RP-HPLC matrix.
21. The process according to claim 1, wherein said chromatographic
stationary phase is a silica or substituted silica material.
22. The process according to claim 21, wherein said chromatographic
stationary phase is C16 or C18 substituted silica.
23. The process according to claim 21, wherein said chromatographic
stationary phase is C4, C8 or phenyl-substituted silica.
24. The process according to claim 21, wherein said chromatographic
stationary phase is a polymeric material.
25. The process according to claim 1, wherein said chromatographic
stationary phase is contacted with said regeneration solution for
at least 1 minute.
26. The process according to claim 1, wherein said chromatographic
stationary phase is contacted with said regeneration solution until
the pressure drop over the length of the chromatographic column at
normal flow rate decreases by at least 10%.
27. The process according to claim 1, wherein contacting of said
chromatographic stationary phase with said regeneration solution is
performed at a temperature in the range from about 5.degree. C. to
50.degree. C.
28. The process according to claim 1, wherein the life time of said
chromatographic stationary phase is at least 500 chromatographic
cycles.
29. The process according to claim 1, wherein said process is
applied to said chromatographic stationary phase for every
chromatographic cycle.
30. The process according to claim 1, wherein said process is
applied to said chromatographic stationary phase at least once
every 100 chromatographic cycles.
31. A process for the production of a therapeutic polypeptide or a
precursor thereof comprising at least one chromatographic step
wherein the chromatographic stationary phase is regenerated by a
process according to claim 1.
32. The process according to claim 31, wherein said therapeutic
polypeptide is a derivative comprising a lipophilic
substituent.
33. The process according to claim 31, wherein said therapeutic
polypeptide is selected from the group consisting of glucagon,
glucagon-like peptide 1, glucagon-like peptide 2, exendin-4, TFF
peptides, human insulin, analogues thereof and derivatives
thereof.
34. The process according to claim 31, wherein said polypeptide or
a precursor thereof is selected from the group consisting of
Lys.sup.26(N.sup..epsilon.-(.gamma.-Glu(N.sup..alpha.-hexadecanoyl)))-GLP-
-1(7-37), Arg.sup.34-GLP-1(7-37), exendin-4,
Lys.sup.17Arg.sup.30-GLP-2(1-33),
Arg.sup.30Lys.sup.17N.sup..epsilon.(.beta.-Ala(N.sup..alpha.-hexadecanoyl-
)) GLP-2(1-33) and Gly.sup.2-GLP-2(1-33).
35. The process according to claim 31, wherein said polypeptide or
a precursor thereof is selected from the group consisting of
threonine methyl ester.sup.B30 human insulin, threonine ethyl
ester.sup.b30 human insulin, Asp.sup.B28 human insulin, threonine
methyl ester.sup.B30 Asp.sup.B28 human insulin, threonine ethyl
ester.sup.B30 Asp.sup.B28 human insulin, Lys.sup.B28 Pro.sup.B29
human insulin, Met.sup.B-1Arg.sup.B0Lys.sup.B28 Pro.sup.B29 human
proinsulin, Lys.sup.B3 Glu.sup.B29 human insulin, Gly.sup.A21
Arg.sup.B31Arg.sup.B32 human insulin, des(B30) human insulin,
N.sup..epsilon.B29-tetradecanoyl des(B30) human insulin,
N.sup..epsilon.B29-litocholoyl-.gamma.-glutamyl des(B30) human
insulin, N.sup..epsilon.B29-octanoyl des(B30) human insulin, and.
N.sup..epsilon.B29-octanoyl human insulin.
36. A chromatographic stationary phase which has been regenerated
by contacting said chromatographic stationary phase with a
regeneration solution comprising at least one organic acid and less
than about 75% w/w water.
37. The chromatographic stationary phase according to claim 36,
wherein said chromatographic stationary phase has been regenerated
by contacting said chromatographic stationary phase with a
regeneration solution comprising at least one organic acid and less
than about 1% w/w water.
38. The chromatographic stationary phase according to claim 36,
wherein said chromatographic stationary phase is a silica or a
substituted silica material.
39. A polypeptide product manufactured by a process comprising the
steps of a) purifying a polypeptide or a precursor thereof using
the chromatographic stationary phase according to claim 36, and b)
isolating said polypeptide or a precursor thereof to give the
resulting polypeptide product.
40. A polypeptide product manufactured by a process wherein is used
a chromatographic stationary phase according to claim 36.
41. An automated chromatographic equipment comprising piping and
control system for implementing the process according to claim
1.
42. A pharmaceutical composition prepared by a process comprising
the steps of a) first purifying a polypeptide or a precursor
thereof using a chromatographic stationary phase regenerated by the
process according to claim 1, b) then drying said polypeptide, and
c) finally admixing with a pharmaceutically acceptable
excipient.
43. A pharmaceutical composition prepared by a process comprising
the steps of a) first purifying a polypeptide or a precursor
thereof using a chromatographic stationary phase regenerated by the
process according to claim 11, b) then drying said polypeptide, and
c) finally admixing with a pharmaceutically acceptable excipient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of international
application no. PCT/DK2004/000234, filed Apr. 2, 2004, which claims
priority to Danish patent application nos. PA 2003 00536, filed
Apr. 8, 2003, and PA 2004 00098 filed Jan. 26, 2004 and U.S. patent
application Nos. 60/462,949, filed Apr. 15, 2003 and 60/539,875
filed Jan. 27, 2004.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of
chromatographic purification. More specifically it pertains to a
process for regenerating chromatographic stationary phases.
BACKGROUND OF THE INVENTION
[0003] Polypeptides are increasingly being used as medicaments for
the treatment of diseases within all major therapy areas. Treatment
of diabetes by chronic insulin administration has been practised
for more than 80 years, and therapeutic applications of
polypeptides within growth disorders and cancer also have been
practised for many years. Economical processes for the large scale
production of polypeptides with a purity sufficiently high for
therapeutic applications are crucial for further polypeptide-based
therapies to reach the mass market and for the existing therapies
to become more widely used.
[0004] Purification of a polypeptide from a mixture is a step which
is normally used several times during the overall manufacturing
process for a therapeutic polypeptide. Reverse phase high pressure
liquid chromatography (RP-HPLC) is the preferred method for
industrial high resolution separation of polypeptides, and the
method has proven versatile for the large scale purification of
many polypeptides.
[0005] Since polypeptides for therapeutic use are to be highly
purified in order not to cause adverse events upon administration
to the patient, it is quite common to use several chromatographic
purification steps in the manufacturing process. The stationary
phase of chromatographic columns in manufacturing plants are
expensive and they are thus used for several chromatographic
cycles. However, over time the performance of the chromatographic
stationary phase declines, i.e. pressure drop over the column
increases prohibitively and the separation factor is impaired. This
has been attributed to the gradual build-up of deposits.
[0006] The problem has been suggested to be overcome by a
regeneration process comprising alkaline buffers (J. Chrom. 461,
1989, 45-61), e.g. pH 7.4 and high concentration of organic
modifier.
[0007] Brange et al. (J. Pharm. Sci. 86 (1997) 517-525) discloses
dissolving insulin fibrils in acid and in base.
[0008] For many years the problem has been alleviated by
regenerating the chromatographic stationary phase with alkaline
solution, e.g. 0.1 molar sodium hydroxide (vide Liliedahl, "Twelve
years of silica-based HPLC purification with focus on peptides", at
Tides 2000, 10 May 2000 in Las Vegas, USA). This regeneration
process may increase the lifetime of silica used for purifying
insulin to between 100 to 600 cycles. However, silica materials are
not stable when exposed to harsh alkaline conditions, and
especially substituted silica materials may not be amenable to
regeneration by alkaline solutions. Economically viable processes
for purifying pharmaceuticals such as therapeutic polypeptides must
include a regeneration process which does not degrade the
chromatographic stationary phase.
[0009] A general and complex process for regenerating particulate
materials (clay, sand, silica etc.) from a wide variety of sources
has been disclosed in WO 00/61493. It is a 5 step process
comprising contacting the material with a) an extractant of organic
material, b) an oxidizing agent, followed by c) an acid solution,
d) heating the material and e) recovering the material. The process
is cumbersome and not amenable for implementation in a
chromatographic purification plant.
[0010] There is a need in the art for more efficient ways of
regenerating chromatographic stationary phases so as to increase
the lifetime of these expensive raw materials and prevent the
pressure drop over chromatographic columns to rise. Especially,
regeneration processes which are suited for in-situ regeneration of
chromatographic stationary phases in manufacturing plants are
needed.
SUMMARY OF THE INVENTION
[0011] The present invention provides a process for regenerating a
chromatographic stationary phase wherein said chromatographic
stationary phase is contacted with a regeneration solution
comprising at least one organic acid and less than about 75% w/w
water.
[0012] In another aspect the present invention provides a process
for regenerating a chromatographic stationary phase wherein said
chromatographic stationary phase is contacted with a regeneration
solution comprising at least one organic acid and less than about
1% w/w water.
[0013] In one embodiment of the invention the organic acid is
formic acid. In another embodiment of the invention the organic
acid is acetic acid. In another embodiment the regeneration
solution contains less than 0.5% water, preferably less than 0.1%
water, more preferably less than 0.02% water and most preferably
less than 0.001% water.
[0014] In another aspect the present invention relates to a process
for regenerating a chromatographic stationary phase wherein said
chromatographic stationary phase is contacted with a regeneration
solution comprising at least one organic acid, an organic solvent
and less than about 1% w/w water.
[0015] In one embodiment of the invention the organic solvent is
ethanol. In another embodiment of the invention the organic solvent
is 2-propanol. In another embodiment of the invention the organic
solvent is acetonitrile. In another embodiment of the invention the
organic solvent is selected from the group consisting of methanol,
1-propanol, and hexylene glycol.
[0016] In another embodiment the regeneration solution contains
less than 0.5% water, preferably less than 0.1% water, more
preferably less than 0.02% water and most preferably less than
0.001% water.
[0017] In another aspect the present invention relates to a
chromatographic stationary phase which has been regenerated by the
processes of the invention.
[0018] In another aspect the present invention relates to a
polypeptide product obtained by the processes of the invention.
[0019] In yet another aspect the present invention relates to a
polypeptide product manufactured by a process comprising the
regeneration of the chromatographic stationary phase by the
processes.
[0020] In yet another aspect the invention relates to an automated
chromatographic equipment comprising piping and control system for
implementing the regeneration process.
[0021] In yet another aspect the present invention relates to a
pharmaceutical composition prepared by purifying a polypeptide
using a chromatographic stationary phase which has been regenerated
by the process.
BREIF DESCRIPTION OF THE DRAWINGS
[0022] The invention is further illustrated in the appended
drawings in which:
[0023] FIG. 1 shows the confocal principle.
[0024] FIG. 2 shows 2D picture of Source 30Q with insulinfibrils
stained with Thioflavin T.
[0025] FIG. 3 shows the principle of the measuring the area of
fibrils on Source 30Q. Light coloured area shows the green light
coming from insulin fibrils on the Source 30Q particles.
[0026] FIGS. 4A-B show preparative chromatograms from
chromatographic purification III (FIG. 4A, upper figure, before
regeneration with formic acid, and FIG. 4B (lower figure) after
regeneration with formic acid).
DEFINITIONS
[0027] The following is a detailed definition of the terms used in
the specification.
[0028] The term "chromatographic stationary phase" as used herein
means the solid phase over which the soluble phase passes, i.e. the
chromatographic matrix. The chromatographic stationary phase is
normally placed within a chromatographic column. Examples of
chromatographic stationary phases are substituted silica, such as
C-4 silica, C-12 silica and C-18 silica, as well as polymeric
materials such as polystyrene, Source 30Q and Sepharose. Additional
examples of chromatographic stationary phases are membranes,
monolithic materials and filters.
[0029] The term "chromatographic eluent" as used herein means the
solution which is used for the elution step where the polypeptide
being purified is normally released from the chromatographic
stationary phase into the eluent. In the normal mode of
chromatography a complete cyclus comprises [0030] a) equilibration
with an equilibration buffer to bring the column in a state where
it is ready for a cyclus, [0031] b) application of the product
holding sample, [0032] c) an optional washing step where the
chromatographic stationary phase with the bound product is washed,
[0033] d) elution where the affinity of the product towards the
chromatography stationary phase decreases and the product leaves
the column in the chromatographic column eluate, and [0034] e) an
optional regeneration where it is attempted to strip the
chromatographic stationary phase from remaining impurities using a
regeneration solution.
[0035] The term "equilibrium buffer" as used herein means the
solution which is used for the equilibration step wherein the
chromatographic column is prepared for a chromatographic cycle.
[0036] The term "regeneration solution" as used herein means a
solution which is used to regenerate a chromatographic stationary
phase. The purpose of the regeneration is keep a satisfactory
performance of the chromatographic separation over several
chromatographic cycles. Typically critical performance related
parameters are the pressure drop over the chromatographic column
and the separation factor. A regeneration step may comprise
contacting of the chromatographic stationary phase with either a
single regeneration solution or with more than one regeneration
solution. In the latter case, each of the regeneration solutions as
well as the resulting mixtures of these are encompassed by the term
"regeneration solution".
[0037] The term "mixture" as used herein means a composition of
matter comprising at least two ingredients. A chromatographic
column eluate is a mixture which comprises the chemicals in the
eluent together with the product which has been stripped from the
column. Another example of a mixture is a solution of a chemical in
a solvent, e.g. saline. Yet another example of a mixture is water
and a water-miscible organic solvent. Yet another example of a
mixture is a solution or suspension of a polypeptide in a solvent
such as water or an organic solvent
[0038] The term "isolating a polypeptide" as used herein means to
bring the polypeptide in a state where it is of higher
concentration or higher purity than it was before isolating it,
i.e. in the starting material. Thus, an example of isolating a
polypeptide is to precipitate or crystallize the polypeptide from a
solution and separate the precipitate or crystals from the mother
liquor.
[0039] The term "organic solvent" as used herein means a solvent
which comprises at least one carbon-atom and which is in the fluid
state throughout the temperature range from 0.degree. C. to
50.degree. C. Non-limiting examples of organic solvents are lower
alcohols such as methanol and ethanol, polyhydric alcohols,
acetonitrile, hexane and acetone.
[0040] The term "water miscible organic solvent" as used herein
means an organic solvent which has a solubility in water at
20.degree. C. of at least 1 g/L. Non-limiting examples of water
miscible organic solvents are ethanol, 1-propanol, 2-propanol,
acetonitrile, and hexyleneglycol.
[0041] The term "organic acid" as used herein means an organic
compound which has at least one functional group with a
dissociation constant, pK.sub.a, of less than 5.0. Examples of
organic acids are formic acid, acetic acid, citric acid etc.
[0042] The term "lower alcohol" as used herein means a
C.sub.1-6-alcohol which is characterized by having between 1 and 6
carbon atoms and one hydroxyl moiety. The carbon skeleton in the
lower alcohol may be straight or branched. Non-limiting examples of
lower alcohols are ethanol, n-propanol, iso-propanol, and
t-butanol.
[0043] The term "polyhydric alcohol" as used herein means an
alcohol having at least two hydroxyl moieties. Non-limiting
examples of polyhydric alcohols are hexylene glycol
(4-methyl-2,4-pentanediol) and neopentyl alcohol
(2,2-dimethyl-1,3-propanediol).
[0044] The term "excipient" as used herein means compounds which
are added to pharmaceutical compositions in order to stabilize and
preserve the composition. Typical excipients are buffers,
preservatives and tonicity modifiers.
[0045] The term "pharmaceutical composition" as used herein means a
product comprising an active compound or a salt thereof together
with pharmaceutical excipients such as buffer, preservative and
tonicity modifier, said pharmaceutical composition being useful for
treating a disease or disorder. Thus a pharmaceutical composition
is also known in the art as a pharmaceutical formulation.
[0046] The term "buffer" as used herein refers to a chemical
compound which is used in a solution to reduce the tendency of pH
of the solution to change over time as would otherwise occur due to
chemical reactions. Buffers include chemicals such as sodium
phosphate, TRIS, glycine and sodium citrate.
[0047] The term "tonicity modifier" as used herein refers to a
chemical compound in a pharmaceutical composition that serves to
modify the osmotic pressure of the pharmaceutical composition so
that the osmotic pressure becomes closer to that of human plasma.
Tonicity modifiers include NaCl, glycerol, D-mannitol etc.
[0048] The term "pharmaceutically acceptable" as used herein means
suited for normal pharmaceutical applications, i.e. does not cause
adverse events in patients etc.
[0049] The term "human insulin" as used herein means the human
hormone whose structure and properties are well known. Human
insulin has two polypeptide chains that are connected by disulphide
bridges between cysteine residues, namely the A-chain and the
B-chain. The A-chain is a 21 amino acid peptide and the B-chain is
a 30 amino acid peptide, the two chains being connected by three
disulphide bridges: one between the cysteines in position 6 and 11
of the A-chain, the second between the cysteine in position 7 of
the A-chain and the cysteine in position 7 of the B-chain, and the
third between the cysteine in position 20 of the A-chain and the
cysteine in position 19 of the B-chain.
[0050] The term "polypeptide" as used herein means a compound
composed of at least ten constituent amino acids connected by
peptide bonds. The constituent amino acids may be from the group of
the amino acids encoded by the genetic code and they may natural
amino acids which are not encoded by the genetic code, as well as
synthetic amino acids. Natural amino acids which are not encoded by
the genetic code are e.g. hydroxyproline, .gamma.-carboxyglutamate,
ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic
amino acids comprise amino acids manufactured by chemical
synthesis, i.e. D-isomers of the amino acids encoded by the genetic
code such as D-alanine and D-leucine, Aib (.alpha.-aminoisobutyric
acid), Abu (.alpha.-aminobutyric acid), Tle (tert-butylglycine);
and .beta.-alanine.
[0051] The term "therapeutic polypeptide" as used herein means a
polypeptide for which there is a recognized potential utility as a
therapeutic agent. Therapeutic polypeptides are typically highly
purified and they are subjected to clinical studies as part of the
regulatory approval process. Examples of therapeutic polypeptides
are human insulin, thrombopoetin, erythropoietin and human growth
hormone.
[0052] The term "polypeptide product" as used herein means a
composition comprising the polypeptide. Examples of polypeptide
products are crystallized polypeptide, precipitated polypeptide,
and a solution of the polypeptide.
[0053] The term "analogue" as used herein in relation to a parent
polypeptide means a modified polypeptide wherein one or more amino
acid residues of the parent polypeptide have been substituted by
other amino acid residues and/or wherein one or more amino acid
residues have been deleted from the parent polypeptide and/or
wherein one or more amino acid residues have been deleted from the
parent polypeptide and/or wherein one or more amino acid residues
have been added to the parent polypeptide. Such addition or
deletion of amino acid residues can take place at the N-terminal of
the polypeptide or at the C-terminal of the polypeptide or within
the polypeptide. An example of an analogue is Arg.sup.34-GLP-1
(7-37) which is a GLP-1(7-37) polypeptide wherein the Lys at
position 34 has been replaced with an Arg. Other examples are
porcine or bovine insulin which are both analogues of human
insulin.
[0054] The term "precursor" as used herein in relation to a
polypeptide means a modified version of the polypeptide which is
being produced. Precursors of a polypeptide are typically amino
acid extended versions of the polypeptide, or truncated versions of
the polypeptide. These precursor may serve to enhance cellular
expression, comprise affinity tags for purification, protect
certain reactive groups of the polypeptide being produced, etc.
[0055] The term "derivative" as used herein in relation to a parent
polypeptide means a chemically modified parent polypeptide or an
analogue thereof, wherein at least one substituent is not present
in the parent polypeptide or an analogue thereof, i.e. a parent
polypeptide which has been covalently modified. Typical
modifications are amides, carbohydrates, alkyl groups, acyl groups,
esters, PEGylations and the like. Examples of derivatives of human
insulin are threonine methyl ester.sup.B30 human insulin and
N.sup..epsilon.B29-tetradecanoyl des(B30) human insulin.
[0056] The term "lipophilic substituent" as used herein means a
substituent comprising 4-40 carbon atoms and having a solubility in
water at 20.degree. C. in the range from about 0.1 mg/100 ml water
to about 250 mg/100 ml water, such as in the range from about 0.3
mg/100 ml water to about 75 mg/100 ml water. For instance, octanoic
acid (C8) has a solubility in water at 20.degree. C. of 68 mg/100
ml, decanoic acid (C10) has a solubility in water at 20.degree. C.
of 15 mg/100 ml, and octadecanoic acid (C18) has a solubility in
water at 20.degree. C. of 0.3 mg/100 ml.
[0057] The term "piping and control system" as used herein means
the physical means (pipes and control valves) and the software
controlling the pipes and valves of a process equipment.
DESCRIPTION OF THE INVENTION
[0058] The present invention is concerned with a process for
regenerating a chromatographic stationary phase wherein said
chromatographic stationary phase is contacted with a regeneration
solution comprising at least one organic acid and less than about
75% w/w water. The present invention is also concerned with a
process for regenerating a chromatographic stationary phase wherein
said chromatographic stationary phase is contacted with a
regeneration solution comprising at least one organic acid and less
than about 1% w/w water.
[0059] The present invention is also concerned with a process for
regenerating a chromatographic stationary phase wherein said
chromatographic stationary phase is contacted with a regeneration
solution having a concentration of organic acid which is at least
25% w/w. A number of organic acids may be used in the regeneration
solution of the process. A preferred organic acid is formic acid.
Another organic acid for the regeneration solution is acetic acid.
Another regeneration solution comprises two organic acids, e.g.
formic acid and acetic acid.
[0060] The regeneration solution may furthermore comprise an
organic solvent. Preferably the organic solvent is also used in the
equilibrium buffer or chromatographic eluent. In one embodiment of
the invention the organic solvent is ethanol. In another embodiment
the organic solvent is 2-propanol. In another embodiment the
organic solvent is acetonitrile. In another embodiment the organic
solvent is selected from the group consisting of methanol,
1-propanol and hexylene glycol.
[0061] In another embodiment, the organic acid is formic acid and
the organic solvent is ethanol. In another embodiment, the organic
acid is formic acid and the organic solvent is acetonitrile. In yet
another embodiment, the organic acid is formic acid and the organic
solvent is 2-propanol. In yet another embodiment, the organic acid
is formic acid and the organic solvent is hexylene glycol. In
another embodiment, the organic acid is acetic acid and the organic
solvent is ethanol. In another embodiment, the organic acid is
acetic acid and the organic solvent is acetonitrile. In yet another
embodiment, the organic acid is acetic acid and the organic solvent
is 2-propanol. In yet another embodiment, the organic acid is
acetic acid and the organic solvent is hexylene glycol.
[0062] In another embodiment, the present invention relates to a
process for regenerating a chromatographic stationary phase wherein
said chromatographic stationary phase is contacted with a
regeneration solution comprising at least one organic acid and less
than 0.5% water, preferably less than 0.1% water, more preferably
less than 0.02% water and most preferably less than 0.001%
water.
[0063] The chromatographic stationary phase is preferably contacted
with the regeneration solution inside the chromatographic column.
In this way a minimum of production capacity is lost due to
down-time in connection with the regeneration step. Thus, the
process of regenerating the chromatographic stationary phase can be
performed without repacking the column. In one embodiment, the
chromatographic stationary phase is fluidized during said
regeneration. In another embodiment the chromatographic eluent or
equilibrium buffer is displaced by a water miscible organic solvent
before said chromatographic stationary phase is contacted with said
regeneration solution. Preferably said water miscible organic
solvent is also present in the chromatographic eluent or
equilibrium buffer. Preferably said water miscible organic solvent
is also present in the regeneration solution.
[0064] In another embodiment the chromatographic stationary phase
is contacted with said regeneration solution outside the
chromatographic column. This procedure is more cumbersome than
performing the regeneration process inside the column, but it may
nevertheless be useful if precipitated material is trapped between
the chromatographic stationary phase particle. In the latter case,
the precipitated material can be removed from the chromatographic
stationary phase without dissolving said material.
[0065] In one embodiment the chromatographic stationary phase is a
RP-HPLC matrix. The chromatographic stationary phase for RP-HPLC
are mechanically very rigid materials which may be silica or
substituted silica such as C4, C6, C8, C10, C12, C16, C18, C30 or
phenyl silica, or it may be a pressure stable polymeric material
which is substituted or unsubstituted. The chromatographic
stationary phase, be it a silica based matrix or a polymeric
material, may also be present in the columns as monolithic rods
with macropores and mesopores.
[0066] Suitable silica material for use as chromatographic
stationary phase is spherical particles with a narrow pore size
distribution and particle sizes in the range from 3 .mu.m to 100
.mu.m, such as from 5 .mu.m to 100 .mu.m, such as from 8 .mu.m to
30 .mu.m, such as 10 .mu.m, 13 .mu.m, 15 .mu.m, 16 .mu.m, 18 .mu.m
and 20 .mu.m. Typically pore sizes in the range of 60 .ANG. to 300
.ANG., such as 100 .ANG., 120 .ANG., 150 .ANG., 175 .ANG., 200
.ANG. or 300 .ANG., are used. For pressure stable polymeric
materials the pore size may be from 10 .ANG. or even higher, e.g.
50 .ANG., 100 .ANG., 400 .ANG., 600 .ANG., 1000 .ANG. or 3000
.ANG.. In one embodiment the pressure stable polymeric material is
Source 30Q or XAD 1180. The chromatographic column is packed with
the stationary phase and after appropriate testing of the quality
of the packing, the column is equilibrated with the buffer used in
the binding mode. Production scale chromatographic columns
typically have diameters of 15 to 100 cm, and such systems may have
dynamic axial compression. For production of small volume
polypeptides the production columns may have a diameter of e.g. 15
cm, 20 cm or 25 cm. For production of large volume polypeptides the
production columns may have a diameter of e.g. 40 cm, 60 cm, 80 cm
or larger.
[0067] In another embodiment of the invention, the chromatographic
stationary phase being regenerated is a membrane, monolithic
materials, filters or the like.
[0068] In one embodiment of the process for regenerating a
chromatographic stationary phase, said chromatographic stationary
phase is contacted with said regeneration solution for at least 1
second, preferably for at least 1 minute, more preferably for at
least 5 minutes, such as from 1 minute to 24 hours, from 1 minute
to 5 hours, from 1 minute to 2 hours, from 10 minutes to 60
minutes.
[0069] In another embodiment of the process for regenerating a
chromatographic stationary phase, said chromatographic stationary
phase is contacted with said regeneration solution until the
pressure drop over the length of the chromatographic column at
normal flow rate decreases by at least 10%, preferably at least
25%, even more preferably at least 50%.
[0070] In another embodiment of the process for regenerating a
chromatographic stationary phase, contacting of said
chromatographic stationary phase with the regeneration solution is
performed at a temperature in the range from about 0.degree. C. to
70.degree. C., from 5.degree. C. to 50.degree. C., such as from
10.degree. C. to 40.degree. C., such as from 15.degree. C. to
30.degree. C., or from 18.degree. C. to 25.degree. C.
[0071] In another embodiment of the process for regenerating a
chromatographic stationary phase, the life time of said
chromatographic stationary phase is at least 500 chromatographic
cycles, preferably at least 700 chromatographic cycles, more
preferably at least 1000 chromatographic cycles, most preferably at
least 2000 chromatographic cycles.
[0072] In another embodiment of the process for regenerating a
chromatographic stationary phase, said process is applied to said
chromatographic stationary phase for every chromatographic cycle,
at least once every 2 chromatographic cycles, at least once every 5
chromatographic cycles, at least once every 20 chromatographic
cycles, at least once every 50 chromatographic cycles, or at least
once every 100 chromatographic cycles.
[0073] In another embodiment of the invention, the number of
regeneration processes performed on a chromatographic stationary
phase is at least 25, at least 50, at least 100, at least 200, at
least 400 or at least 1000.
[0074] In another embodiment of the process for regenerating a
chromatographic stationary phase, said process is applied to said
chromatographic stationary phase whenever the pressure drop over
the length of the chromatographic column exceeds a threshold
value.
[0075] Another aspect of the present invention is a process for the
production of a therapeutic polypeptide or a precursor thereof,
said process comprising at least one chromatographic step wherein
the chromatographic stationary phase is regenerated by a
regeneration process as described above. In one embodiment of the
process for the production of a therapeutic polypeptide or a
precursor thereof, said therapeutic polypeptide is a derivative
comprising a lipophilic substituent. In another embodiment of the
process for the production of a therapeutic polypeptide or a
precursor thereof, said therapeutic polypeptide is a derivative
comprising a lipophilic substituent attached to the .epsilon.-amino
group of a lysine residue. In another embodiment of the process for
the production of a therapeutic polypeptide or a precursor thereof,
said therapeutic polypeptide is selected from the group consisting
of glucagon, glucagon-like peptide 1, glucagon-like peptide 2,
exendin-4, TFF peptides, human insulin, analogues thereof and
derivatives thereof. In another embodiment said polypeptide is
selected from the group consisting of
Lys.sup.26(N.sup..epsilon.-(.gamma.-Glu(N.sup..alpha.-hexadecanoyl)))-GLP-
-1(7-37), Arg.sup.34-GLP-1(7-37), exendin-4, Lys
.sup.17Arg.sup.30-GLP-2(1-33), Arg.sup.30Lys.sup.17
N.sup..epsilon.(.beta.-Ala(N.sup..alpha.-hexadecanoyl)) GLP-2(1-33)
and Gly.sup.2-GLP-2(1-33). In another embodiment said polypeptide
is exendin-4. In another embodiment said polypeptide is a fusion
polypeptide comprising human serum albumin or a fragment thereof.
In another embodiment said polypeptide is a fusion polypeptide
between GLP-1(7-37) or an analogue thereof and a human serum
albumin fragment or an analogue thereof. In another embodiment said
polypeptide is a fusion polypeptide between exendin-4(1-39) or an
analogue thereof and a human serum albumin fragment or an analogue
thereof. In another embodiment said polypeptide is a fusion
polypeptide comprising the Fc portion of an immunoglobulin or a
fragment thereof. In another embodiment said polypeptide is a
fusion polypeptide between GLP-1 (7-37) or an analogue thereof and
a fragment of the Fc portion of an immunoglobulin or an analogue
thereof. In another embodiment said polypeptide is a fusion
polypeptide between exendin-4(1-39) or an analogue thereof and a
fragment of the Fc portion of an immunoglobulin or an analogue
thereof.
[0076] In another embodiment said polypeptide is selected from the
group consisting of human insulin, a human insulin precursor, a
human insulin analog, a human insulin analog precursor, a
GLP-1(7-37) analogue, an exendin-4(1-39) analogue, and derivatives
thereof. In another embodiment said polypeptide is selected from a
human insulin derivative comprising at least one methoxy or ethoxy
moiety. In another embodiment said polypeptide is selected from the
group consisting of [0077] threonine methyl ester.sup.B30 human
insulin, [0078] threonine ethyl ester.sup.B30 human insulin, [0079]
Asp.sup.B28 human insulin, [0080] threonine methyl ester.sup.B30
Asp.sup.B28 human insulin, [0081] threonine ethyl ester.sup.B30
Asp.sup.B28 human insulin, [0082] LyS.sup.B28 Pro.sup.B29 human
insulin, [0083] Met.sup.B-1Arg.sup.B0Lys.sup.B28 Pro.sup.B29 human
proinsulin, [0084] Lys.sup.B3 Glu.sup.B29 human insulin, [0085]
Gly.sup.A21 Arg.sup.B31 Arg.sup.B32 human insulin, [0086] des(B30)
human insulin, [0087] N.sup..epsilon.B29-tetradecanoyl des(B30)
human insulin, [0088]
N.sup..epsilon.B29-litocholoyl-.gamma.-glutamyl des(B30) human
insulin, [0089] N.sup..epsilon.B29-octanoyl des(B30) human insulin,
and. [0090] N.sup..epsilon.B29-octanoyl human insulin.
[0091] In yet another embodiment said polypeptide is selected from
human serum albumin, erythropoietin, TNF-.alpha., an interleukin,
IGF-1, IGF-2, human growth hormone, somatostatin, human amylin and
analogues thereof.
[0092] The polypeptides being purified on chromatographic
stationary phases regenerated by the processes of the present
invention may be produced by a variety of techniques known in the
art of polypeptide production. Polypeptides larger than 3000 Dalton
are usually produced by fermentation or cell culture, whereas
smaller polypeptide may be produced by chemical peptide synthesis.
Other important factors determining the optimal production method
are also the amount of polypeptide to be produced and the structure
of the polypeptide, e.g. disulphide bonds and other modifications.
Fermentation or cell culture derived polypeptides are commonly
produced by cultivation of recombinant host cells, e.g. bacteria,
fungi mammalian cells, insect cells or plant cells in appropriate
cultivation media. The cultivation medium may be a more or less
chemically defined medium containing the necessary nutrients for
growth and product formation of the host cells, e.g. sugar,
nitrogen source, salts, vitamins and other growth factors. Once the
microorganisms or the cells have been cultivated in the medium and
they have optionally been disrupted, the cultivation medium
contains the desired product in a mixture with remnant medium
components, host cell derived impurities and product related
impurities. Host cell derived impurities are mainly polypeptides,
nucleic acids and cellular debris. The product is separated from
these non-related impurities in the recovery or early purification
steps. In the final purification steps (polishing) where impurities
closely related to the product polypeptide are separated from the
product polypeptide, chromatographic steps are extensively
used.
[0093] Synthesis of polypeptides may also be performed via solid
phase synthesis by Merrifield-type chemistry, by solution phase
methods, or by semisynthetic methods known in the art.
[0094] One or more chemical conversion steps may be performed
in-between the recovery and the final purification steps. Such
chemical modifications may by the hydrolysis of a precursor
polypeptide wherein the amino acid extension on the polypeptide is
cleaved of the polypeptide. Such amino acid extensions may be used
for increasing the host cell expression in the case of culture
derived polypeptides, or it may be used to specifically purify the
polypeptide, such as by affinity chromatography e.g. IMAC
purification of histidine tagged polypeptides. The chemical
conversion can also be the chemical modification to produce a
polypeptide which is a derivative, e.g. by acylation, PEGylation or
esterification. Such chemical modifications are well known in the
art (see e.g. WO 98/08871, WO 99/43706, U.S. Pat. No. 5,424,286, WO
00/09666, WO 00/66629, WO 01/04156 and WO 02/90388).
[0095] Another aspect of the present invention is the use of the
above processes for regenerating a chromatographic stationary phase
for decreasing the pressure drop over the length of the
chromatographic column.
[0096] Another aspect of the present invention is the use of the
above processes for regenerating a chromatographic stationary phase
for the manufacture of a therapeutic polypeptide.
[0097] Another aspect of the present invention is a chromatographic
stationary phase which has been regenerated by contacting said
chromatographic stationary phase with a regeneration solution, said
regeneration solution comprising at least one organic acid and less
than about 75% w/w water. Another aspect of the present invention
is a chromatographic stationary phase which has been regenerated by
contacting said chromatographic stationary phase with a
regeneration solution, said regeneration solution comprising at
least one organic acid and less than about 1% w/w water.
[0098] In one embodiment the chromatographic stationary phase has
been regenerated by a process as described above. In another
embodiment the chromatographic stationary phase has been
regenerated by a process, wherein said regeneration solution
contains less than 0.5% water, preferably less than 0.1% water,
more preferably less than 0.02% water and most preferably less than
0.001% water. In a further embodiment the regenerated
chromatographic stationary phase is a silica, or a substituted
silica material.
[0099] In another aspect the present invention relates to a
polypeptide product manufactured by a process comprising the steps
of [0100] a) purifying a polypeptide or a precursor thereof using
the chromatographic stationary phase produced by the regeneration
process of the present invention, and [0101] b) isolating said
polypeptide or a precursor thereof to give the resulting
polypeptide product.
[0102] In another aspect the present invention relates to a
polypeptide product manufactured by a process wherein is used a
chromatographic stationary phase regenerated according to the
process of the present invention.
[0103] In another aspect the present invention relates to an
automated chromatographic equipment comprising piping and control
system for implementing the regeneration process according to the
present invention.
[0104] In another aspect the present invention relates to a
pharmaceutical composition prepared by a process comprising the
steps of [0105] a) first purifying a polypeptide or a precursor
thereof using a chromatographic stationary phase regenerated by the
process according to the present invention, [0106] b) then drying
said polypeptide, and [0107] c) finally admixing with a
pharmaceutically acceptable excipient.
[0108] In another aspect the present invention relates to a
pharmaceutical composition prepared by a process comprising the
steps of [0109] a) first purifying a polypeptide or a precursor
thereof using a chromatographic stationary phase regenerated by the
process wherein said chromatographic stationary phase is contacted
with a regeneration solution comprising at least one organic acid
and less than 0.5% water, preferably less than 0.1% water, more
preferably less than 0.02% water and most preferably less than
0.001% water, and [0110] b) then drying said polypeptide, and
[0111] c) finally admixing with a pharmaceutically acceptable
excipient.
EXAMPLES
[0112] The following acronyms for commercially available chemicals
and materials are used: [0113] HCOOH: Formic acid [0114] EtOH:
Ethanol [0115] AcOH: Acetic acid [0116] ODDMS: Octadecyldimethyl
substituted silica particles (ODDMS silica)
[0117] The formic acid used for examples 1-68, 70-71, 73-74 and 76
has a specified purity of 98-100% (according to manufacturer), and
the formic acid used for example 72 has a purity of 99.9%.
[0118] The abbreviation CV means Column Volumes as known in the
field of chromatography.
Example 1-68
[0119] The over all set-up of the experiments of example 1-68 was
as follows: [0120] 1) Determination of the back pressure and height
of valley of a column with no pressure problems (a new unused
column). [0121] 2) Introduction of pressure and performance
problems. [0122] 3) Determination of the back pressure and height
of valley of a column with pressure problems. [0123] 4)
Regeneration of the column. [0124] 5) Determination of the back
pressure and height of valley of a column after regeneration.
Determination of the Back Pressure and Height of Valley:
[0125] The same type of silica gel was used for all the experiments
mentioned in this section. The silica gel is an ODDMS 200 .ANG., 15
.mu.m silica gel. Mostly, the same gel batch (batch no. 205144) was
used (all the columns starting with 874-).
[0126] The silica gel was packed in 10 mm.times.250 mm steel
columns and tested in a functionality test using DesB30 insulin as
the test substance and eluting with water-ethanol mixtures
containing calcium chloride and potassium chloride salts (see table
1). The back pressure under elution and the height of valley
between DesB30 insulin and the nearest impurity (in front of the
insulin peak) were used as the test parameters for how well the
column had regained its performance.
[0127] The elution time of DesB30insulin may be subject to some
experimental variation due to small variations in temperature and
other experimental parameters. When the elution time of
DesB30insulin is the same in different experiments the height of
valley is a perfect comparison of the columns separation
performance. When the elution time of DesB30insulin varies between
experiments the height of vallay is a less good measure of the
column separation performance. All else equal, the longer the
retention time is the lower the height of valley will be.
TABLE-US-00001 TABLE 1 Solutions used in the functionality test.
Inlet Buffer Type Content (w/w) A11 1 Equlibration buffer 20%
ethanol A12 2 Elution buffer A 25% ethanol, 1.5% KCl, 0.4%
CaCl.sub.2 and 0.15% triethanol amine (pH 7.4 with HCl) B1 3
Elution buffer B 35% ethanol, 1.5% KCl, 0.4% CaCl.sub.2 and 0.15%
triethanol amine (pH 7.4 with HCl) A13 4 Regeneration 70% ethanol
and 6.9% acetic acid buffer A18 Application DesB30 insulin
Na.sub.2EDTA solution, pH 7.5, ethanol
[0128] The functionality test was performed at 23.+-.2.degree. C.
on an Aktaexplorer 100A with Unicorn 4.0 as the control software
and running the following column cycle (se table 2): TABLE-US-00002
TABLE 2 Column cycle in the functionality test (CV is column
volumes). Action CV (no.) Volume (ml) Equilibration 3 58.9 Loading
0.8 15 Wash 1 1 19.6 Elution 10 196.3 Wash 2 0.5 9.8 Rinse 2
39.3
[0129] A column packed with silica gel (batch 205144) was tested
and the back pressure measured to 3.4.+-.0.1 MPa. Similarly the
back pressure was determined for other columns before introducing
pressure problems. Experiments show that if these values are
regained after treatment, the column has regained its
performance.
[0130] The height of valley was also determined for the individual
columns before and after introduction of pressure and performance
problems, and after regeneration of the column.
Introduction of Pressure and Performance Problems:
[0131] Pressure and performance problems were introduced in 1 of 3
ways: [0132] 1) The column was used in the functionality test but
was taken off the system after loading and placed at 70.degree. C.
for 1-16 hours. Thereafter the back pressure and height of valley
were measured performing the rest of the functionality test. [0133]
2) The ODDMS silica gel (25 g) was stirred in a beaker with DesB30
insulin (0.14 g), 0.1 M Tris buffer (22 ml) and ethanol (15 ml) at
50.degree. C. for 1 hour. Thereafter the gel was decanted and
packed in a steel column (10 mm.times.250 mm). The backpressure and
height of valley were measured performing the functionality test.
This method can also be performed at lower temperatures but that
demands longer reaction times. [0134] 3) A combination of 1) and
2). Firstly, the gel was treated as in 2) but after packing, the
column was treated as in 1).
[0135] The methods used for the individual columns are listed in
table 4 below.
Regeneration of the Column (e.i. Removal of Pressure and
Performance Problems):
[0136] The regeneration of the column was also performed on the
Aktaexplorer 100. The column cycle for this process can be seen in
table 3 below. After regeneration, the column was again tested in
the functionality test and the back pressure determined.
TABLE-US-00003 TABLE 3 Column cycle under regeneration. Action
Solvent CV (no.) Volume (ml) Wash 1 EtOH 3 58.9 Regeneration See
table 4 and 5 2 39.3 Left to stand -- 0 30 minutes Regeneration See
table 4 and 5 2 39.3 Wash 2 EtOH 3 58.9
[0137] Different solvents were testet at 22.degree. C. and
40.degree. C. (the entire HPLC system was placed in a refrigerator
at the chosen temperature .+-.1.degree. C.). The specific
conditions for the individual columns are shown in table 4 and the
results from the functionality tests are shown in table 5.
[0138] Specific Experimental Conditions for the Individual Columns:
TABLE-US-00004 TABLE 4 Experimental conditions for individual
columns. Ex- Introduction Tem- am- of problems pera- ple Column by
ture no. no. method no. Regeneration solvent (.degree. C.) 1
204377/1 1 HCOOH 22 2 874-32/1 2 HCOOH 22 3 874-32/2 3 HCOOH 22 4
874-32/7 2 HCOOH 22 5 874-33/9 2 HCOOH 22 6 874-33/6 3 HCOOH 22 7
874-24/22 2 HCOOH 40 8 874-24/10 3 AcOH 22 9 874-24/15 2 AcOH 22 10
874-24/23 2 AcOH 40 11 874-31/1 2 AcOH 40 12 203635/5 1 HCOOH/AcOH
99:1 22 13 874-24/6 3 HCOOH/AcOH 99:1 22 14 205019/1 1 HCOOH/AcOH
99:1 40 15 204770/1 1 HCOOH/AcOH 3:1 22 16 874-24/26 2 HCOOH/AcOH
3:1 40 17 204888/1 1 HCOOH/AcOH 1:1 22 18 874-24/27 2 HCOOH/AcOH
1:1 40 19 204923/1 1 HCOOH/AcOH 1:3 22 20 874-24/28 2 HCOOH/AcOH
1:3 40 21 203251/3 1 HCOOH/EtOH 99:1 22 22 874-24/4 2 HCOOH/EtOH
99:1 22 23 204815/1 1 HCOOH/EtOH 99:1 40 24 204815/1 1 HCOOH/EtOH
3:1 22 25 204888/1 1 HCOOH/EtOH 3:1 40 26 874-24/5 3 HCOOH/EtOH 1:1
22 27 874-24/16 2 HCOOH/EtOH 1:1 22 28 204923/1 1 HCOOH/EtOH 1:1 40
29 204550/1 1 HCOOH/EtOH 1:3 22 30 874-24/17 2 HCOOH/EtOH 1:3 22 31
204770/1 1 HCOOH/EtOH 1:3 40 32 203635/5 1 HCOOH/water 99:1 22 33
874-24/24 2 HCOOH/water 99:1 40 34 204377/1 1 HCOOH/water 95:5 22
35 874-24/29 2 HCOOH/water 95:5 40 36 202985/2 1 HCOOH/water 4:1 22
37 874-24/30 2 HCOOH/water 4:1 40 38 204550/1 1 HCOOH/water 1:1 22
39 874-24/31 2 HCOOH/water 1:1 40 40 874-33/5 3 HCOOH/water 1:1 40
41 874-24/7 3 HCOOH/water 1:3 22 42 874-24/18 2 HCOOH/water 1:3 22
43 204770/1 1 HCOOH/water 1:3 40 44 874-24/8 3 AcOH/water 99:1 22
45 874-24/19 2 AcOH/water 99:1 40 46 874-24/11 3 AcOH/water 3:1 22
47 874-24/20 2 AcOH/water 3:1 40 48 874-31/2 2 AcOH/water 3:1 40 49
874-33/4 3 AcOH/water 3:1 40 50 874-24/12 3 AcOH/water 1:1 22 51
874-24/25 2 AcOH/water 1:1 40 52 874-31/3 2 AcOH/water 1:1 40 53
874-24/13 3 HCOOH/phenol/water 22 1:1:1 54 874-24/21 2
HCOOH/phenol/water 22 1:1:1 55 874-32/3 2 1500 ppm Na- 22
hypochlorite 56 874-33/11 3 1500 ppm Na- 22 hypochlorite 57
874-33/14 2 1500 ppm Na- 22 hypochlorite 58 874-32/4 2 Performic
acid 22 59 874-33/13 3 Performic acid 22 60 874-33/18 2 Performic
acid 22 61 874-32/5 2 1.5M formaldehyde 22 62 874-33/12 3 1.5M
formaldehyde 22 63 874-33/15 2 1.5M formaldehyde 22 64 874-32/6 2
6M guanidine 22 hydrochloride 65 874-33/10 3 6M guanidine
hydrochloride 22 66 874-33/17 2 6M guanidine hydrochloride 22 67
874-33/19 2 EtOH (60% w/w)/0.1M 22 NaOH 68 874-33/20 3 EtOH (60%
w/w)/0.1M 22 NaOH
[0139] Functionality Test Results for the Individual Columns:
TABLE-US-00005 TABLE 5 Back pressure and height of valley results
from functionality tests performed before and after introduction of
problems and after regeneration of the columns according to the
conditions listed in table 4. RT is the retention time for DesB30
insulin and height of valley (HV) is for DesB30 insulin and the
nearest impurity. Performance Before After After Back pressure
(MPa) problems problems regeneration Example Before After HV RT HV
RT HV no. problems problems After regeneration RT ml mAU ml mAU ml
mAU 1 3.8 6.2 3.8 140.2 38.4 144.3 36.1 2 3.4 5.8 3.6 141.2 39.6
146.6 62.0 138.6 44.5 3 3.4 >10 3.7 141.2 39.6 -- -- 137.9 45.9
4 3.4 4.4 3.4 141.2 39.6 146.6 62.0 139.0 43.2 5 3.4 4.7 3.4 141.7
41.9 134.6 63.0 139.2 42.9 6 3.4 >10 3.6 141.7 41.9 -- -- 140.1
45.8 7 3.4 4.7 3.4 134.5 54.3 136.5 47.8 137.2 48.6 8 3.4 >10
>10 134.5 54.3 -- -- -- -- 9 3.4 4.7 4.4 134.5 54.3 136.5 47.8
135.9 48.4 10 3.4 4.7 3.9 134.5 54.3 136.5 47.8 135.0 50.1 11 3.4
4.2 4.2 134.5 54.3 149.5 44.7 12 3.0 3.7 2.8 136.1 42.7 130.3 51.9
13 3.4 >10 3.5 134.5 54.3 -- -- 145.2 44.4 14 3.5 >10 3.5
139.9 44.1 -- -- 135.9 44.8 15 3.5 >10 3.3 138.7 40.6 -- --
127.3 54.7 16 3.4 4.7 3.4 134.5 54.3 136.5 47.8 139.3 45.0 17 3.0
>10 2.9 139.2 30.4 -- -- 130.2 36.2 18 3.4 4.7 3.4 134.5 54.3
136.5 47.8 137.1 47.0 19 3.1 >10 3.0 130.2 36.2 -- -- 133.3 57.2
20 3.4 4.7 3.5 134.5 54.3 136.5 47.8 136.6 47.5 21 2.2 3.4 2.3
131.6 32.9 133.8 35.2 22 3.4 5.2 3.4 134.5 54.3 136.5 47.8 135.1
50.1 23 3.4 5.8 3.3 148.5 29.6 142.0 65.0 24 3.3 6.2 3.1 135.2 35.4
135.3 34.0 25 3.1 9 3.1 157.0 24.7 -- -- 151.3 31.9 26 3.4 >10
>10 134.5 54.3 -- -- -- -- 27 3.4 4.7 3.5 134.5 54.3 136.5 47.8
136.2 46.9 28 3.2 >10 3.5 139.7 44.7 -- -- 148.0 35.7 29 2.8
>10 >10 142.4 23.2 -- -- -- -- 30 3.4 4.7 4.1 134.5 54.3
136.5 47.8 137.6 44.2 31 3.6 >10 >10 147.4 38.7 -- -- -- --
32 3.1 6.9 3.1 143.8 34.7 136.3 41.4 33 3.4 4.7 3.3 134.5 54.3
136.5 47.8 132.1 55.8 34 3.7 >10 3.9 140.6 41.6 -- -- 145.8 34.5
35 3.4 4.7 3.6 134.5 54.3 136.5 47.8 139.8 42.0 36 2.4 5.1 2.3
145.8 22.0 134.1 32.0 37 3.4 4.7 3.5 134.5 54.3 136.5 47.8 140.3
45.4 38 2.6 4.6 2.7 126.6 27.2 135.9 34.5 39 3.4 4.7 3.4 134.5 54.3
136.5 47.8 140.4 42.2 40 3.4 >10 4.1 141.7 41.9 -- -- 141.8 64.0
41 3.4 >10 >10 134.5 54.3 -- -- -- -- 42 3.4 4.7 3.3 134.5
54.3 136.5 47.8 137.0 45.6 43 3.4 5.8 3.8 131.9 47.0 145.0 40.8 44
3.4 >10 >10 134.5 54.3 -- -- -- -- 45 3.4 4.7 4.3 134.5 54.3
136.5 47.8 135.2 42.9 46 3.4 >10 5.2 134.5 54.3 -- -- 134.2
119.3 47 3.4 4.7 3.4 134.5 54.3 136.5 47.8 134.4 64.0 48 3.4 4.2
3.5 134.5 54.3 137.3 47.7 49 3.4 >10 5.6 141.7 41.9 -- -- 141.9
73.4 50 3.4 >10 9 134.5 54.3 136.5 47.8 -- -- 51 3.4 4.7 3.4
134.5 54.3 136.5 47.8 134.9 50.7 52 3.4 4.2 3.5 134.5 54.3 126.9
62.7 53 3.4 >10 4.1 134.5 54.3 -- -- 137.7 46.1 54 3.4 4.7 3.4
134.5 54.3 136.5 47.8 138.6 45.0 55 3.4 4.4 3.8 141.2 39.6 146.6
62.0 138.0 46.2 56 3.4 >10 >10 141.7 41.9 -- -- -- -- 57 3.4
4.7 4.1 141.7 41.9 134.6 63.0 149.8 39.3 58 3.4 4.4 3.6 141.2 39.6
146.6 62.0 138.2 46.1 59 3.4 >10 3.5 141.7 41.9 -- -- 135.5 52.3
60 3.4 4.7 3.3 141.7 41.9 134.6 63.0 135.1 54.1 61 3.4 4.4 3.7
141.2 39.6 146.6 62.0 137.7 49.4 62 3.4 >10 >10 141.7 41.9 --
-- -- -- 63 3.4 4.7 3.9 141.7 41.9 134.6 63.0 147.6 40.7 64 3.4 4.4
3.5 141.2 39.6 146.6 62.0 139.4 45.0 65 3.4 >10 >10 141.7
41.9 -- -- -- -- 66 3.4 4.7 3.8 141.7 41.9 134.6 63.0 137.4 53.4 67
3.4 4.7 3.5 141.7 41.9 134.6 63.0 138.4 39.1 68 3.4 >10 3.9
141.7 41.9 134.6 -- 138.8 49.3
Example 69
Column Life Time--Chromatographic Solvents/Eluents.
[0140] The life time of substituted silica gels may be limited by
the chemical degradations of the gel by the solutions applied to
the chromatographic column, e.g. buffers and regeneration
solutions. Chemical degradation of substituted silica gels may be
observed by the appearance of silicium in the exit from the column,
or by lowering of the carbon contents of the gel showing loss of
the substitution. The following experiments show the silicium
appearance in column effluent and the decrease of the carbon
contents of the gels during prolonged flushing with three
chromatographic solutions: 20% ethanol in water, eluent 1 and
eluent 2. Composition of eluent 2 is 31% w/w ethanol, 1.5% w/w KCl,
0.40% w/w CaCl.sub.2, 0.15% w/w triethanol amine and pH adjusted to
pH 7.4 using HCl. The composition of eluent 1 is salt, buffer,
ethanol and pH 3.0.
[0141] A batch of ODDMS silica gel was packed in five 4.0.times.250
mm steel columns by standard procedure for packing chromatographic
columns. The columns were then equilibrated with 3 CV of ethanol
before continuous flushing with a chromatographic eluent was
started. The five columns were then flushed with a chromatographic
eluent (20% EtOH, eluent 1 or eluent 2) for 1, 3, 7, 12, and 16
days, respectively. After the appropriate flushing time each column
were then equilibrated with 3 CV of ethanol and the silica gel was
taken out of the columns. A sample of the spent silica gel was
subjected to analysis of the carbon contents, and a sample of the
spent regeneration solution was subjected to analysis for the
silicium contents. The results for day no 0 is the result (carbon
contents) of the silica gel used for packing the columns.
TABLE-US-00006 TABLE 6 Assessment of column lifetime during
prolonged flushing with typical chromatographic eluents by
measurement of remaining carbon and released silicium. Measured
Column Duration Measured C Si Volume Relative C no (days) (%) (mg)
(mL) (%) Chrom solution 100 .ANG. 0 19.40 0.00 0 100.00 ODDMS
204614/15 20% EtOH 1 19.14 0.05 136 98.66 4.0 * 250 mm 204614/16
20% EtOH 3 19.16 0.13 425 98.76 204614/17 20% EtOH 7 19.26 0.31
1540 99.28 204614/23 20% EtOH 12 19.28 0.25 1270 99.38 204614/24
20% EtOH 16 19.27 0.41 1360 99.33 0 19.40 0.00 0 100.00 204614/18
Eluent 1 1 18.67 0.15 147 96.24 204614/20 Eluent 1 3 18.85 0.36 360
97.16 204614/21 Eluent 1 7 18.76 0.87 870 96.70 204614/19 Eluent 1
12 18.64 1.33 1325 96.08 204614/22 Eluent 1 16 18.67 0.86 860 96.24
20% EtOH (% v/v) 200 .ANG. 0 9.80 0.00 0 100.00 ODDMS 204630/14 20%
EtOH 1 9.61 0.03 160 98.06 4.0 * 250 mm 204630/13 20% EtOH 3 9.66
0.09 430 98.57 204630/15 20% EtOH 7 9.72 0.09 425 99.18 204630/16
20% EtOH 12 9.69 0.18 610 98.88 204630/18 20% EtOH 16 9.69 0.28 930
98.88 0 9.80 0.00 0 100.00 204630/19 Eluent 2 1 9.55 0.13 125 97.45
204630/17 Eluent 2 3 9.55 0.40 400 97.45 204630/20 Eluent 2 7 9.54
0.85 850 97.35 204630/21 Eluent 2 12 9.59 1.29 1290 97.86 204630/23
Eluent 2 16 9.49 0.70 700 96.84
Example 70
Column Life Time--Regeneration Solutions Comprising Formic
Acid.
[0142] The life time of substituted silica gels may be limited by
the chemical degradations of the gel by the solutions applied to
the chromatographic column, e.g. buffers and regeneration
solutions. Chemical degradation of substituted silica gels may be
observed by the appearance of silicium in the exit from the column,
and by lowering of the carbon contents of the gel showing loss of
the substitution. The following experiments show the silicium
appearance in column effluent and the decrease of the carbon
contents of the gels during prolonged flushing with formic acid
containing regeneration solutions. Thus, comparing to the situation
using chromatographic buffers or solvents, the formic acid
containing regeneration solutions are not more deteriorating on the
column matrix.
[0143] A batch of ODDMS silica gel was packed in five 4.0.times.250
mm steel columns by standard procedure for packing chromatographic
columns. Each column were then equilibrated with 3 CV 100% EtOH
before continuous flushing with formic acid was started. The five
columns were then flushed with a regeneration solvent being formic
acid (100% or 80%) for 1, 3, 7, 12, and 16 days, respectively.
After the appropriate flushing time each column were then
equilibrated with 3 CV of ethanol and the silica gel was taken out
of the columns. A sample of the spent silica gel was subjected to
analysis of the carbon contents, and a sample of the spent
regeneration solution was subjected to analysis for the silicium
contents. The results for day no 0 is the result (carbon contents)
of the silica gel used for packing the columns. TABLE-US-00007
TABLE 7 Assessment of column lifetime during prolonged regeneration
with solutions containing formic acid by measurement of remaining
carbon and released silicium. Formic Measured Column acid Duration
Measured C Si Volume Relative C Column type no. (%) (days) (%) (mg)
(mL) (%) 100 .ANG. ODDMS 0 -- 0 19.40 0.00 0 100.00 4.0 * 250 mm
204614/7 100 1 19.00 0.82 124 97.94 204614/5 100 3 18.90 1.46 510
97.42 204614/6 100 7 19.26 1.95 620 99.28 204614/3 100 12 18.97
5.63 1430 97.78 204614/2 100 12 18.94 3.92 1100 97.63 0 -- 0 19.40
0.00 0 100.00 204614/8 80 1 19.08 1.67 177 98.35 204614/10 80 3
19.08 2.62 410 98.35 204614/9 80 7 18.93 4.55 970 97.58 204614/11
80 12 17.04 6.07 1080 N.D. 204614/12 80 16 18.61 6.86 2090 95.93
200 .ANG. ODDMS 0 -- 0 9.80 0.00 0 100.00 4.0 * 250 mm 204630/8 100
1 9.56 0.62 85 97.55 204630/5 100 3 9.50 1.77 510 96.94 204630/6
100 7 9.41 1.02 800 96.02 204630/3 100 12 9.67 1.31 1020 98.67
204630/2 100 12 9.63 1.22 1655 98.27 0 -- 0 9.80 0.00 0 100.00
204630/7 80 1 9.64 0.56 141 98.37 204630/10 80 3 9.42 0.77 440
96.12 204630/9 80 7 9.43 1.10 920 96.22 204630/11 80 12 8.67 1.40
1470 N.D. 204630/12 80 16 9.18 1.46 2000 93.67
Example 71
Using Confocal Laser Scanning Microscopy Detecting Insulinfibrils
on Source 30Q Anion Exchanger
Purpose
[0144] The purpose of using Confocal laser scanning microscopy is
to visual determine how effectively different regenerating solvents
are in removing insulin fibrils from Source 30Q. With sophisticated
image processing software allowing the opportunity to measure the
area of insulinfibrils on each picture giving a more precise
judgment in stead of an visual judgment of each picture
The Confocal Principle
[0145] The principle in using CLSM is that the sample is stained
with a fluorescent dye. The stained sample is placed under the
microscope and the fluorescent dye is exitated using a laser. The
emitted light coming from the fluorescent dye is detected and the
image formed.
[0146] Confocal laser scannings microscope is as normal confocal
microscope using a laser as ligthsource. The light from the laser
is going through the objective of the microscope via the
dichromatic beamsplitter. The dichromatic beamsplitter is a device
allowing the light from the laser to go through the objective down
to the sample and the emitted light coming from the sample to pass
the dichromatic beamsplitter and go up to the photomultiplier tubes
where the light is detected and the image is formed. Between the
dichromatic beamsplitter and photomultipliertubes is the confocal
pinhole placed. The confocal pinhole works as a filter stopping all
the light coming from out of focus regions in reaching the
photomultipliertubes. This means that the image there is formed
comes from a very narrow focus plane. By adjusting the confocal
pinhole it is possible to move the image focus plane up and down
through the sample and produce a series of images along the optical
(z) axis of the microscope. This series of images can be collected
into 3-D image of the sample.
Source 30Q
[0147] Source 30Q is a strong anion exchanger based on monodisperse
beads with a particle size of 30 micron. The matrix consists of
polystyrene/divinyl benzene. Source 30Q gives a weak
autofluorescence's in the wavelength area above 650 nm. This means
that is possible to see the Source 30Q particles without any
staining.
Thioflavin T
[0148] The dye used for staining insulin fibrils is Thioflavin T
wich is known for staining amyloid proteins. Thioflavin T has a
broad emission spectrum with max emission ranging from below 490 nm
and above 530 nm. This means that it is possible to distinct
between light coming from the insulinfibrils (green) and the
autofluorescence's light coming from the particles (red).
Method for Staining Insulinfibrils on Source 30Q
0.0385 g dry Source 30Q is put into 25-30 ml glass.
500 .mu.l 96% ethanol is added to the Source 30Q
4.5 ml 0.05 M acetic acid adjusted to pH 3 with NaOH is added.
The mixture is mixed together by shaking the glass carefully
20 .mu.l 116.62 mM Thioflavin T in Milli-Q-water is added
The mixture is carefully shaken for 5 min.
The sample is now stained and ready for analysing under the
microscope
A small droplet of the mixture approximately 40 .mu.l I placed in a
microscopic well and put in the microscope
10 2D pictures of each sample from different places of the well was
taken
Equipment
Microscopic conditions
Objective: 40*; oil; NA?
Laser: Argon 488 nm
Laser intensity: 100%
Confocal pinhole: 20 .mu.m
Emission filter (No 2): 515/30M from Chroma
Emission filter (No 3): HQ650LP from Chroma
Microscope: Nikon TE300 equipped with PCM2000 confocal scanhead
(photomultiplier) from Nikon.
Software: Nikon EZ2000 Viewer 2.5.77
Software used for image processing: AnalySIS 3.00
Image Processing
[0149] The area of insulinfibrils was measured on the 10 2D
pictures fro each sample. To ensure that each sample was treated
exactly the same way the image processing was done automatically by
forming a macro. The macro is shown below. The area of each
individual fibril from the 10 pictures of each sample was
summarized in excel worksheet. The summarized fibril area from each
sample was collected in a bar chart for comparison.
Macro for Measuring Area of Fibrils:
[0150] Op. Display=1; [0151] docActivate("picture name"); [0152]
DbLoadlmage( ); [0153] MaximizeContrast( ); [0154]
ShadingCorrection(N*N average filter size; 3; lower limit; 1900;
upper limit; 2000); [0155] BinarzeColorImage(ColorThresholds:=NULL,
Phase:=-1); [0156] Invert( ); [0157] EdgeEnhance(size; 5; percent;
70); [0158] SetFrame(Left:=0, Top:=0, Right:=1023, Bottom:=1023);
[0159] Detect( ); [0160] ParticleResults( ); [0161] Op.Display=6;
[0162] Option. BurnOverlay=TRUE; [0163] SaveAs(FileName:); [0164]
docActivate("Sheet*"); [0165] SaveAs(FileName:);
[0166] Close(AskFor Save:=TRUE); TABLE-US-00008 TABLE 8 Measured
area of fibrils on a spent Source 30Q gel before and after
regeneration of the gel using different regeneration solutions.
Area of fibrils Regeneration solution (random units) None 485 None
458 NaOAc (0.25% w/w NaOAc, 0.24% w/w tris, 154 42.5% w/w EtOH, pH
7.5 NaOH (1M) 101 HCOOH:water (50:50) 68 HCOOH (100%) 0 HCOOH:EtOH
(50:50) 0
[0167] Complete removal of the insulin fibrils are obtained using
pure formic acid or a mixture of formic acid and ethanol. A 50:50
mixture of formic acid:water does not completely eliminate the
fibrils, although this mixture is a more efficient regeneration
solution than sodium hydroxide (1M).
Example 72
[0168] Standard procedure for regeneration of RP-HPLC columns with
formic acid in production scale.
[0169] The procedures described below applies to two types of
columns used in four chromatographic purification steps in a
production scale purification of human insulin.
Type 1 Column:
Bed height 32.5 cm
[0170] 1. The column is flushed with approximately 1.5 CV 20% w/w
ethanol in water with a flow rate of 4.6 CV/h in order to remove
buffer salts [0171] 2. The flow direction in the column is reversed
[0172] 3. The column is flushed with approximately 1.1 CV absolute
ethanol with a flow rate of 4.5 CV/h [0173] 4. 1.1 CV pure formic
acid is pumped onto the column at a flow rate of 2.1 CV/h [0174] 5.
The flow is stopped and the column is allowed to stay with formic
acid for 30 minutes [0175] 6. The column is flushed with
approximately 1.1 CV absolute ethanol with a flow rate of 4.5 CV/h
[0176] 7. The column is flushed with approximately 1.1 CV 20% w/w
ethanol in water with a flow rate of 4.5 CV/h [0177] 8. The flow
direction in the column is switched back to normal [0178] 9. The
column is equilibrated with minimum 4 CV of 20% w/w ethanol in
water with a flow rate of 4.6 CV/h The column is now ready for use
again. Type 2 Column: Bed height 37.5 cm [0179] 1. The column is
flushed with approximately 1.5 CV 20% w/w ethanol in water with a
flow rate of 4.6 CV/h in order to remove buffer salts [0180] 2. The
flow direction in the column is reversed [0181] 3. The column is
flushed with approximately 1.0 CV absolute ethanol with a flow rate
of 3.9 CV/h [0182] 4. 0.92 CV pure formic acid is pumped onto the
column at a flow rate of 1.9 CV/h [0183] 5. The flow is stopped and
the column is allowed to stay with formic acid for 30 minutes
[0184] 6. The column is flushed with approximately 1.0 CV absolute
ethanol with a flow rate of 3.9 CV/h [0185] 7. The column is
flushed with approximately 1.0 CV 20% w/w ethanol in water with a
flow rate of 3.9 CV/h [0186] 8. The flow direction in the column is
switched back to normal [0187] 9. The column is equilibrated with
minimum 4.0 CV of 20% w/w ethanol in water with a flow rate of 4.6
CV/h The column is now ready for use again.
[0188] On the Type 1 columns the regeneration with formic acid is
carried out after every 16.sup.th run as a preventive action to
stop build up of components, increasing column back pressures and
increasing pool volumes. By applying the regeneration procedure
with formic acid in the chromatographic purification I step, the
life time of the column bed is extended up to 2000 runs.
[0189] On the Type 2 columns regeneration with formic acid is
carried out routinely after every 60.sup.th run or if increasing
back pressures, increasing pool volumes and build up of impurities
is observed. By applying the regeneration procedure with formic
acid the life time of the column bed is extended up to 600 runs in
chromatographic purification III step and up to 900 runs in
chromatographic step IV.
[0190] The column matrix used in chromatograhic purification I, III
and IV steps consists of octadecyidimethyl substituted silica
particles (ODDMS silica). The standard procedure described applies
for all types of substituted silica matrixes used in production
scale.
[0191] The procedure is also used in formic acid regeneration
procedures for column matrices based on Source Q material
[0192] Examples of the effect at chromatographic purification III
and IV are shown below. TABLE-US-00009 TABLE 9 Chromatographic
purification III: Effect on pool volume and back pressure. Back
pressure (max. pressure over delivery pump) Before regeneration
with formic acid (100%) Pool volume (weight) 208 kg 27 Bar After
regeneration with formic acid (100%) Pool volume 165 kg 17 Bar
[0193] The regeneration reduces the pool volume with app. 21% and
the back pressure with 37%.
[0194] FIG. 4A-B shows the effect of regeneration on preparative
chromatogram for Chromatographic purification III.
[0195] By applying the formic acid regeneration an improved
separation and human insulin peak form is achieved (insulin peak
being the large peak starting at time 8.20 in upper figure and at
time 14.02 in lower figure).
Example 73
Regeneration of DEAE Sepharose.
[0196] A batch of DEAE Sepharose which had been used for a large
number of purification cycles in a process for purification of
human growth hormone was regenerated by pure formic acid (100%
HCOOH). Before regeneration the Sepharose has a brown colour
indicating deposited material on the gel. The regenerated Sepharose
was much lighter in colour.
[0197] Samples of the regenerated Sepharose was compared to the
Sepharose before regeneration using a colorimeter (Minolta CR-300)
to quantify the colour differences, cn.f. results in table 10.
TABLE-US-00010 TABLE 10 Assessment of the regeneration of DEAE
Sepharose when regenerated as a slurry with a regeneration solution
of formic acid. Colour coordinates Sepharose material analysed L
(light) A (red) b (yellow) Spent gel before regeneration 68.81 2.37
17.22 Spent gel after 3 hour regeneration 84.99 0.66 11.55 Spent
gel after 24 hour regeneration 87.94 0.35 7.64 Spent gel after 24
hour regeneration 85.85 0.69 8.81 where fresh regeneration solution
has been applied at 12 hour. Unspent gel, i.e. which has never
86.44 -0.06 2.01 been used for chromatography
Example 74
Regeneration of Spent Silica Gel Containing Glucagon
Aggregates.
[0198] Glucagon and the glucagon-like peptides (GLP-1 and GLP-2)
are particularly susceptible to aggregation where they form
fibrils, i.e. aggregates of .beta.-sheet structures. In this
example a model experiment was carried out where human glucagon was
loaded onto a silica column (as described in examples 1-68). The
glucagon solution was allowed to stay in the column for 3 days at
30.degree. C. in order to introduce the pressure and performance
problems mimicking the problems encountered during the industrial
manufacture of glucagon. The pressure over the column was measured
to 3.5 MPa using eluent 2 as described in example 69 at a flow rate
of 9 mL/min at 22.degree. C.
[0199] Following a regeneration cycle using formic acid (100%) the
pressure over the column was reduced to 2.67 MPa illustrating the
effectiveness of formic acid as a regeneration solution.
Example 75
Column Life Time--Regeneration Solution Containing 0.1 M NaOH and
60% w/w Ethanol in Water.
[0200] The experiment was carried out as described in example 70
except for the regeneration solution which in this experiment was
alkaline ethanol as commonly used to regenerate chromatographic
stationary phases.
[0201] The flow of regeneration solution was approximately 0.1
mL/min, and the experiments were terminated after 4 days as the
pressure over the columns were >10 MPa. The results until break
down of the columns are shown in table 11. TABLE-US-00011 TABLE 11
Assessment of column life time during prolonged regeneration with a
regeneration solution containing 0.1M NaOH and 60% w/w ethanol in
water. Meas- Meas- Rela- Dura- ured ured Vol- tive Column tion C Si
ume C Column type no. (days) (%) (mg) (mL) (%) 200 .ANG. ODDMS 0 0
9.80 0.0 0 100 4.0 * 250 mm 204630/26 1 9.80 16.2 116 100 204630/25
2 9.77 27.0 180 99.7 204630/24 4 9.59 31.9 228 97.9
Example 76
Regeneration of XAD 1180
[0202] The lifetime of the reverse phase polymeric resin XAD 1180
used for concentrating of an insulin precursor from clarified
fermentation broth was limited because of severe accumulation of
hydrophobic contaminants such as colored compounds, peptides and
antifoams from the fermentation. The normal regeneration cycle was
based on washing with a regeneration solution containing 80%
Ethanol in 0.1M citric acid, pH 3.0 followed by heating with a 5%
NaOH solution at 80.degree. C. This regeneration process was not
efficient to remove accumulated contaminants.
[0203] Removal of bound antifoam (Pluronic PE 6100 and P2000) from
spent resin was evaluated by experiments with different
concentration and contacting time of ethanol and isopropanol in
static mode and compared to a formic acid regeneration (Table 12).
The formic acid treatment is superior in removing the adsorbed
contaminants compared to standard industrial regeneration solvents.
TABLE-US-00012 TABLE 12 Efficiency of different regeneration
solutions in removing adsorbed antifoam contaminants from a spent
XAD 1180 chromatographic stationary phase. Conc. Regeneration Time
Temp. of antifoam on solvent Conc. (hours) (.degree. C.) resin
(ppm) Ethanol 84% 2 25 20023 Ethanol 92% 2 25 13058 Ethanol 99% 2
25 8042 Ethanol 84% 6 25 25083 Ethanol 84% 2 50 14712 Ethanol 99% 6
50 9911 Isopropanol 70% 2 25 15899 Isopropanol 99% 2 25 3262 Formic
acid 99% 2 25 670
* * * * *