U.S. patent application number 10/978110 was filed with the patent office on 2009-01-08 for pulmonary insulin crystals.
Invention is credited to Svend Havelund.
Application Number | 20090010854 10/978110 |
Document ID | / |
Family ID | 27220590 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090010854 |
Kind Code |
A9 |
Havelund; Svend |
January 8, 2009 |
Pulmonary insulin crystals
Abstract
The present invention provides methods and compositions for
treating diabetes by administering acylated insulin or an acylated
insulin analog via a pulmonary route. The insulin or insulin analog
may be in the form of a dry powder or a solution.
Inventors: |
Havelund; Svend; (Bagavaerd,
DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
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Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050118114 A1 |
June 2, 2005 |
|
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Family ID: |
27220590 |
Appl. No.: |
10/978110 |
Filed: |
October 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10152535 |
May 20, 2002 |
6818738 |
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10978110 |
Oct 29, 2004 |
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09836496 |
Apr 17, 2001 |
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10152535 |
May 20, 2002 |
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09045038 |
Mar 20, 1998 |
6310038 |
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09836496 |
Apr 17, 2001 |
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60041390 |
Mar 27, 1997 |
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Current U.S.
Class: |
424/46 ;
514/5.9 |
Current CPC
Class: |
A61K 9/145 20130101;
A61K 38/28 20130101; A61K 9/0075 20130101; C07K 14/62 20130101 |
Class at
Publication: |
424/046 ;
514/003 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61L 9/04 20060101 A61L009/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 1997 |
DK |
0317/97 |
Claims
1. A method of treating diabetes in a diabetic patient, the method
comprising the steps of: a. recovering from a solution a zinc free
insulin crystals having a diameter in below 10 microns when
recovered from solution; b. providing the crystals to a patient for
inhalation; and c. instructing the patient to inhale the
crystals.
2. The method of claim 1, wherein the insulin crystals that are
recovered have an as recovered diameter in the range below 5
microns.
3. The method of claims 1 or 2, wherein the recovered crystals are
crystals of a rapid acting analog insulin.
4. The method of claim 3, wherein the insulin is a Lys Pro human
insulin.
5. The method of claims 1 or 2 further comprising combining the
insulin crystals with an enhancer.
6. The method of claim 3, further comprising combing the Lys Pro
crystals with an enhancer.
7. The method of claim 1, further comprising, after step c,
dispersing the insulin crystals with a compressed gas to form an
aerosol further comprising the step of flowing the aerosol into the
patient's respiratory track
8. A method of treating diabetes in a diabetic patient, the method
comprising the steps of: a. providing a diabetic patient with an
apparatus for inhaling powdered insulin comprised of insulin
crystals that have a diameter of less than 10 microns; b. providing
insulin crystals having a diameter of less than 10 microns, wherein
the crystals are recovered from a solution and wherein the crystals
as recovered have a diameter of less than 10 microns; c.
aerosolizing the insulin crystals within the apparatus provided in
step a; d. flowing the aerosolized insulin crystals to the
patient's respiratory track.
9. A method of treating a diabetic patient to reduce blood glucose
levels within the patient, the method comprising the steps of: a.
obtaining a solution containing insulin; b. recovering from the
solution a zinc free insulin crystal that when recovered has a
diameter of less than 10 microns; c. providing the zinc free
crystal to a diabetic patient; d. instructing the patient to inhale
the crystal to reduce blood glucose levels.
10. A plurality of insulin crystal comprising zinc free insulin
crystals wherein the crystals have not been machined or milled and
wherein the crystals have been recovered from a solution and
wherein at least some of the crystals have an as recovered diameter
of less than 10 microns.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/152,535 filed on May 20, 2002 and claims priority under 35
U.S.C. 119 of Danish application no. 0317/97 filed on Mar. 20,
1997, of U.S. provisional application No. 60/041,390 filed on Mar.
27, 1997 and the benefit of application Ser. Nos. 09/045,038,
09/836,496 and 10/152,535 filed on Mar. 20, 1998, Apr. 17, 2001 and
May 20, 2002 respectively, in the U.S. is claimed under 35 U.S.C.
120, the contents of all of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to zinc free insulin crystals
having a diameter below 10 .mu.m and to therapeutic powder
formulations suitable for pulmonary administration comprising such
insulin crystals.
BACKGROUND OF THE INVENTION
[0003] Diabetes is a general term for disorders in man having
excessive urine excretion as in diabetes mellitus and diabetes
insipidus. Diabetes mellitus is a metabolic disorder in which the
ability to utilize glucose is more or less completely lost. About
2% of all people suffer from diabetes.
[0004] Since the introduction of insulin in the 1920's, continuous
strides have been made to improve the treatment of diabetes
mellitus. To help avoid extreme glycaemia levels, diabetic patients
often practice multiple injection therapy, whereby insulin is
administered with each meal.
[0005] Insulin is usually administrated by s.c. or i.m. injections.
However, due to the adherent discomfort of injections alternative
ways of administration such as nasal and pulmonary has been
extensively investigated. For a review on alternative routes of
administration of insulin, see Danielsen et al. New routes and
means of insulin delivery, in: Childhood and Adolescent Diabetes
(Ed. Kelnar), Chapman & Hall Medical, London 1994, pp.
571-584.
[0006] In order to circumvent injections, administration of insulin
via the pulmonary route could be an alternative way to provide
absorption profiles which mimic the endogenous insulin without the
need to inject the insulin.
DESCRIPTION OF THE BACKGROUND ART
[0007] Administration of insulin via the pulmonary route can be
accomplished by either an aqueous solution or a powder preparation.
A description of the details can be found in several references,
one of the latest being by Niven, Crit. Rev. Ther. Drug Carrier
Sys, 12(2&3): 151-231 (1995). One aspect covered in said review
is the stability issue of protein formulations, aqueous solutions
being less stable than powder formulation. So far, all powder
formulations have been described as mainly amorphous.
[0008] A review of the permeation enhancers useful for the
promotion of trans-mucosal absorption is found in Sayani et al.,
Crit. Rev. Ther. Drug Carrier Sys, 13(1&2): 85-184 (1996).
[0009] Patton et al., Inhale Therapeutic Systems, PCT WO 95/24183,
claim a method for aerosolising a dose of insulin comprising
providing the insulin as a dry powder dispersing an amount of the
dry powder in a gas stream to form an aerosol capturing the aerosol
in a chamber for subsequent inhalation.
[0010] It has been found that when insulin is combined with an
appropriate absorption enhancer and is introduced into the lower
respiratory tract in the form of a powder of appropriate particle
size, it readily enters the systemic circulation by absorption
through the layer of epithelial cells in the lower respiratory
tract as described in U.S. Pat. No. 5,506,203. The manufacturing
process described in said patent, comprising dissolution of insulin
at acid pH followed by a pH adjustment to pH 7.4 and addition of
sodium taurocholate before drying the solution by vacuum
concentration, open drying, spray drying, or freeze drying, results
in a powder composed of human insulin and absorption enhancer. The
powder is characterized as mainly amorphous determined under a
polarized light microscope. The desired particle size distribution
is achieved by micronizing in a suitable mill, such as a jet mill,
and the components may be mixed before or after micronizing. The
biological effect of the powder obtained according to the methods
described in this patent is only seen in the presence of a
substantial amount of enhancer.
[0011] Platz et al., Inhale Therapeutic Systems, PCT WO 96/32149,
describes spray drying of zinc insulin from a solution containing
mannitol and a citrate buffer, pH 6.7. The inlet temperature is 120
to 122.quadrature.C, the outlet temperature 80-81.quadrature.C. The
mass median aerodynamic diameter, MMAd, of the obtained insulin
particles was determined to 1.3 to 1.5 .mu.m.
[0012] In his thesis, "Insulin Crystals", Munksgaard Publisher
1958, p. 54-55, Schlichtkrull describes crystallisation of zinc
free, recrystallised porcine insulin from a solution comprising
0.01 M sodium acetate and 0.7% .about.0.12 M sodium chloride and
0.1% methyl-parahydroxybenzoate and using a pH of 7.0. The crystals
obtained were 10-50 .mu.m rhombic dodecahedral crystals showing no
birefringence.
[0013] Jackson, U.S. Pat. No. 3,719,655 describes a method of
purification of crude porcine and bovine insulin by
crystallisation. Zinc free crystals of insulin are obtained by
crystallisation at pH 8.2 (range 7.2-10) in the presence of 0.5 M
(range 0.2 M-1 M) of a sodium, potassium, lithium or ammonium salt.
Crystallisation is achieved by addition of 1 N alkali metal
hydroxide or 1 N ammonia to a solution of crude insulin in 0.5 N
acetic acid to a pH of 8.2 is obtained. Alternatively,
crystallisation is achieved in an aqueous solution of impure
insulin at pH 8.2 by addition of solid sodium chloride to a
concentration of sodium ions of 0.45 M. The crystals appear in the
octadecahedral or dodecahedral forms, i.e. crystals belonging to
the cubic crystal system.
[0014] Baker et al., Lilly, EP 0 709 395 A2 (filed Oct. 31, 1994)
describe a zinc free crystallisation process for
Lys.sup.B28-Pro.sup.B29 human insulin characterised by adjustment
of the pH of a strongly buffered acid solution containing metal
cations or ammonium ions and a preservative with metal hydroxide or
ammonia to a value between 8.5 and 9.5.
[0015] The known methods for the manufacture of insulin particles
of the desired size for pulmonary administration are cumbersome,
generates problems with insulin dust and the investments in
equipment are large. Furthermore, insulin is exposed to conditions
where some denaturation is likely to take place. Thus WO 96/32149
disclose spray drying in a temperature range of 50.quadrature.C to
100.quadrature.C, followed by milling of the particles to achieve
to desired particle size.
[0016] Furthermore, the known powder formulations for pulmonary
administration which have been described as mainly amorphous have a
tendency to associate into aggregates in the dry powder.
DESCRIPTION OF THE INVENTION
Definitions
[0017] The expression "enhancer" as used herein refers to a
substance enhancing the absorption of insulin, insulin analogue or
insulin derivative through the layer of epithelial cells lining the
alveoli of the lung into the adjacent pulmonary vasculature, i.e.
the amount of insulin absorbed into the systemic system is higher
than the amount absorbed in the absence of enhancer.
[0018] In the present context the expression "powder" refers to a
collection of essentially dry particles, i.e. the moisture content
being below about 10% by weight, preferably below 6% by weight, and
most preferably below 4% by weight.
[0019] The diameter of the crystals is defined as the Martin's
diameter. It is measured as the length of the line, parallel to the
ocular scale, that divides the randomly oriented crystals into two
equal projected areas
BRIEF DESCRIPTIONS OF THE INVENTION
[0020] It is an object of the present invention to provide an
insulin powder suitable for pulmonary delivery which has a reduced
tendency to associate into aggregates in the dry powder compared to
the pulmonary insulin particles described in the prior art.
[0021] According to the present invention this object has been
accomplished by providing zinc free insulin crystals having a
diameter below 10 .mu.m.
[0022] The crystals of the present invention furthermore exhibit a
better stability profile than powders of essentially the same
composition prepared by spray drying, freeze-drying, vacuum drying
and open drying. This is probably due to the amorphous state of
powders prepared by the other methods described. By this means it
is possible to store the powder formulations based on the crystals
of the present invention at room temperature in contrary to human
insulin preparations for injections and some amorphous insulin
powders without stabilizing agent which have to be stored between
2.quadrature.C to 8.quadrature.C.
[0023] Furthermore, therapeutical powder formulations comprising
the insulin crystals of the invention elucidates better flowing
properties than corresponding amorphous powder formulations.
Preferred Embodiments
[0024] The zinc free insulin crystals of the invention are
advantageously provided in a crystal structure belonging to the
cubic crystal system, preferably in the octadecahedral or
dodecahedral crystal forms, since these crystal forms result in
readily soluble product having excellent flowing properties.
[0025] The diameter of the insulin crystals is advantageously kept
in the range of 0.2 to 5 .mu.m, preferably in the range of 0.2 to 2
.mu.m, more preferably in the range of 0.5 and 1 .mu.m, to ensure
high bioavailability and suitable profile of action, see PCT
application No. WO 95/24183 and PCT application No. WO
96/32149.
[0026] In a preferred embodiment the insulin used is selected from
the group consisting of human insulin, bovine insulin or porcine
insulin, preferably human insulin.
[0027] In another preferred embodiment the insulin used is selected
from the group consisting of rapid-acting insulins, preferably
des(B30) human insulin, Asp.sup.B28 human insulin or
Lys.sup.B28Pro.sup.B29 human insulin.
[0028] In another preferred embodiment the insulin used is an
insulin derivative, preferably selected from the group consisting
of B29-N.sup..epsilon.-myristoyl-des(B30) human insulin,
B29-N.sup..epsilon.-palmitoyl-des(B30) human insulin,
B29-N.sup..epsilon.-myristoyl human insulin,
B29-N.sup..epsilon.-palmitoyl human insulin,
B28-N.sup..epsilon.-myristoyl Lys.sup.B28Pro.sup.B29 human insulin,
B28-N.sup..epsilon.-palmitoyl Lys.sup.B28Pro.sup.B29 human insulin,
B30-N.sup..epsilon.-myristoyl-Thr.sup.B29Lys.sup.B30 human insulin,
B30-N.sup..epsilon.-palmitoyl-Thr.sup.B29Lys.sup.B30 human insulin,
B29-N.sup..epsilon.-(N-palmitoyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(N-lithocholyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(.omega.-carboxyheptadecanoyl)-des(B30) human
insulin and B29-N.sup..epsilon.-(.omega.-carboxyheptadecanoyl)
human insulin, more preferably Lys.sup.B29(N-.epsilon. acylated)
des(B30) human insulin.
[0029] The insulin derivatives has a protracted onset of action and
may thus compensate the very rapid increase in plasma insulin
normally associated with pulmonary delivery. By carefully selecting
the type of insulin, the present invention enables adjustment of
the timing and to obtain the desired biological response within a
defined time span.
[0030] In order to avoid irritation of the lungs and to eliminate
immunological reactions, the employed insulin is preferably insulin
which has been purified by chromatography, such as MC insulin
(Novo), Single Peak insulin (E. Lilly) and RI insulin
(Nordisk).
[0031] In a preferred embodiment the zinc free insulin crystals
according to the invention further comprise a stabilizing amount of
a phenolic compound, preferably m-cresol or phenol, or a mixture of
these compounds.
[0032] The present invention is furthermore concerned with a
therapeutic powder formulation suitable for pulmonary
administration comprising the zinc free crystals described
above.
[0033] In a preferred embodiment this therapeutic powder
formulation further comprises an enhancer which enhances the
absorption of insulin in the lower respiratory tract.
[0034] The enhancer is advantageously a surfactant, preferably
selected from the group consisting of salts of fatty acids, bile
salts or phospholipids, more preferably a bile salt.
[0035] Preferred fatty acids salts are salts of C.sub.10-14 fatty
acids, such as sodium caprate, sodium laurate and sodium
myristate.
[0036] Lysophosphatidylcholine is a preferred phospholipid.
[0037] Preferred bile salts are salts of ursodeoxycholate,
taurocholate, glycocholate and taurodihydrofusidate. Still more
preferred are powder formulations according to the invention
wherein the enhancer is a salt of taurocholate, preferably sodium
taurocholate.
[0038] The molar ratio of insulin to enhancer in the powder
formulation of the present invention is preferably 9:1 to 1:9, more
preferably between 5:1 to 1:5, and still more preferably between
3:1 to 1:3.
[0039] The powder formulations of the present invention may
optionally be combined with a carrier or excipient generally
accepted as suitable for pulmonary administration. The purpose of
adding a carrier or excipient may be as a bulking agent,
stabilizing agent or an agent improving the flowing properties.
[0040] Suitable carrier agents include 1) carbohydrates, e.g.
monosaccharides such as fructose, galactose, glucose, sorbose, and
the like; 2) disaccharides, such as lactose, trehalose and the
like; 3) polysaccharides, such as raffinose, maltodextrins,
dextrans, and the like; 4) alditols, such as mannitol, xylitol, and
the like; 5) inorganic salts, such as sodium chloride, and the
like; 6) organic salts, such as sodium citrate, sodium ascorbate,
and the like. A preferred group of carriers includes trehalose,
raffinose, mannitol, sorbitol, xylitol, inositol, sucrose, sodium
chloride and sodium citrate.
[0041] The crystals of the present invention are advantageously
produced according to the following procedure: [0042] providing a
solution of insulin having a pH between 7.0 and 9.5; [0043] mixing
said solution with a solution of a salt of an alkali metal or an
ammonium salt; and [0044] recovering the formed crystals.
[0045] The salt of an alkali metal or ammonium is preferably
selected from the group consisting of the hydrochloride or acetate
of sodium, potassium, lithium or ammonia, or mixtures thereof, more
preferably sodium acetate.
[0046] In order to suppress the solubility of the crystals formed,
the solution of insulin and/or the solution of a salt of an alkali
metal or an ammonium salt preferably comprises a water miscible
organic solvent in an amount which corresponds to 5 to 25% (v/v) in
the solution obtained after mixing.
[0047] The water miscible organic solvent is preferably selected
from the group consisting of ethanol, methanol, acetone and
2-propanol, more preferably ethanol.
[0048] A very uniform distribution of crystal sizes and crystals of
the same crystallographic form are obtained when the two solutions
are mixed within a period of less than 2 hours, preferably less
than 1 hour, more preferably less than 15 minutes, still more
preferably less than 5 minutes.
[0049] The crystallisation process by which uniformly sized, small,
zinc free crystals is obtained directly, without the use of
milling, micronizing, sieving and other dust generating steps, is
much to be preferred from the present state of the art in the
manufacture of insulin powders for inhalation.
[0050] The concentration of insulin after mixing is preferably
between 0.5% and 10%, more preferably between 0.5% and 5%, still
more preferably between 0.5% and 2%.
[0051] The concentration of salt after mixing is preferably between
0.2 M and 2 M, more preferably about 1 M.
[0052] The method according to the present invention may further
comprise a washing step, in which the crystals obtained are washed
with a solution comprising auxiliary substances to be included in
the final dry powder, preferably an enhancer and/or a carbohydrate,
and optionally comprising 5-25% of an alcohol, preferably ethanol,
5-50 mM of a preservative preferably phenol, and 0.1-2 M of a salt
such as sodium acetate.
[0053] This invention is further illustrated by the following
examples which, however, are not to be construed as limiting.
EXAMPLE 1
Crystallisation in 1 M Sodium Acetate.
[0054] 2 g of highly purified human insulin is dissolved in 100 ml
10 mM tris buffer, pH 8.0 in 20% (v/v) of ethanol in water. To this
solution is added 100 ml 2 M sodium acetate under stirring. A
precipitate forms immediately. After 2 days at room temperature
microscopy shows small crystals having a diameter between 0.5 and 1
.mu.m. The crystals are collected by centrifugation at
-10.quadrature.C, washed once with 20 ml ice cold 10% ethanol (v/v)
in water, isolated by centrifugation and dried by lyophilization.
The obtained crystals are shown in FIG. 1.
EXAMPLE 2
Crystallisation in the Presence of Taurocholic Acid Sodium
Salt.
[0055] 10 mg of human insulin and 5 mg of taurocholic acid sodium
salt are dissolved in 500 .mu.l 10 mM tris buffer, pH 8.0 in 20%
(v/v) of ethanol in water. To this solution is added 500 .mu.l 2 M
sodium acetate. Microscopy after 1 hour and after 24 hours shows
identically appearance of the crystals, i.e. uniformly sized
crystals having diameters between 0.5 and 1 .mu.m. The crystals
were washed three times with 100 .mu.l 10% (v/v) ethanol in water
at -10.quadrature.C and dried in vacuo. HPLC of the crystals showed
that the washings had removed the taurocholic acid sodium salt from
the insulin crystals.
EXAMPLE 3
Crystallisation in the Presence of Tween 80, bis(2-ethylhexyl)
Sulfosuccinate Sodium Salt, Chitosan,
L-.alpha.-lysophosphatidylcholine Myristoyl and Polyoxyethylene
Sorbitan Monolaurate.
[0056] Crystallisation was performed as described in Example 2
except that taurocholic acid sodium salt was replaced by 0.6% (w/v)
Tween 80, 0.56% (w/v) bis(2-ethylhexyl) sulfosuccinate sodium salt,
0.32% (w/v) chitosan, 0.52% (w/v) L-.alpha.lysophosphtidylcholine
myristoyl, and 1% (w/v) polyoxyethylene sorbitan monolaurate,
respectively. All five examples resulted in uniformly sized
crystals having diameters between 0.5 and 1 .mu.m.
EXAMPLE 4
[0057] Crystallisation in 10% (v/v) Ethanol.
[0058] Crystallisation was performed in 10% (v/v) ethanol as
described in Example 1, using 4 combinations of pH and
concentration of sodium acetate: [0059] 4.1: pH 7.5 and 1 M sodium
acetate [0060] 4.2: pH 7.5 and 1.5 M sodium acetate [0061] 4.3: pH
9.0 and 1 M sodium acetate [0062] 4.4: pH 9.0 and 1.5 M sodium
acetate
[0063] All 4 combinations yielded uniformly sized crystals having
diameters between 0.5 and 1 .mu.m.
EXAMPLE 5
Crystallisation in 15% (v/v) Ethanol.
[0064] Crystallisation was performed in 15% (v/v) ethanol, using 6
combinations of pH and concentration of sodium acetate: [0065] 5.1:
pH 7.5 and 1 M sodium acetate [0066] 5.2: pH 7.5 and 1.5 M sodium
acetate [0067] 5.3: pH 7.5 and 2 M sodium acetate [0068] 5.4: pH
9.0 and 1 M sodium acetate [0069] 5.5: pH 9.0 and 1.5 M sodium
acetate [0070] 5.6: pH 9.0 and 2 M sodium acetate
[0071] All 6 combinations yielded uniformly sized crystals having
diameters between 0.5 and 1 .mu.m.
EXAMPLE 6
Crystallisation in 20% (v/v) Ethanol.
[0072] Crystallisation was performed in 20% (v/v) ethanol using 4
combinations of pH and concentration of sodium acetate: [0073] 6.1:
pH 7.5 and 1 M sodium acetate [0074] 6.2: pH 7.5 and 1.5 M sodium
acetate [0075] 6.3: pH 7.5 and 2 M sodium acetate [0076] 6.4: pH
9.0 and 1 M sodium acetate
[0077] All 4 combinations yielded uniformly sized crystals having
diameters between 0.5 and 1 .mu.m.
EXAMPLE 7
Crystallisation at pH 7.5, 8.0, 8.5 and 9.0 in 20% Ethanol (v/v)
Using Slow Addition of Sodium Acetate.
[0078] Crystallisation was performed using solutions as described
in Example 1, except that the 2 M sodium acetate was dissolved in
20% (v/v) ethanol in water. The pH of the insulin solutions were
adjusted to 7.5, 8.0, 8.5 and 9.0, respectively. The sodium acetate
solution was added in aliquots over a period of 2 hours, using 10
min between additions. At all 4 pH values uniformly sized crystals
having diameters between 0.5 and 1 .mu.m were obtained.
EXAMPLE 8
Crystallisation of Lys.sup.B29(.epsilon.-myristoyl) des(B30) Human
Insulin in the Presence of Taurocholic Acid Sodium Salt.
[0079] 10 mg of Lys.sup.B29(.epsilon.-myristoyl) des(B30) human
insulin and 5 mg of taurocholic acid sodium salt are dissolved in
500 .mu.l 10 mM tris buffer, pH 8.0 in 20% (v/v) of ethanol in
water. To this solution is added 500 .mu.l 2 M sodium acetate.
Microscopy after 1 hour and after 24 hours shows identically
appearance of the crystals, i.e. uniformly sized crystals having
diameters between 0.5 and 1 .mu.m. The crystals were washed once
with 300 .mu.l 10% (v/v) ethanol in water at -10.quadrature.C and
dried in vacuo. HPLC of the crystals showed that the washings had
removed the palmitoyl-Thr.sup.B29Lys.sup.B30 human insulin,
B29-N.sup..epsilon.-(N-palmitoyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(N-lithocholyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(.omega.-carboxyheptadecanoyl)-des(B30) human
insulin and B29-N.sup..epsilon.-(.omega.-carboxyheptadecanoyl)
human insulin.
* * * * *