FIELD OF THE INVENTION
The present invention relates to monomeric analogs of human
insulin. More specifically, the present invention relates to
various parenteral formulations, which comprise a monomeric
insulin analog, zinc, protamine, and a phenolic derivative.
The formulations provide a prolonged duration of action. A
process for preparing insulin analog-protamine formulations
is also described.
BACKGROUND OF THE INVENTION
Since the introduction of insulin in the 1920's, continuous
strides have been made to improve the treatment of diabetes
mellitus. Major advances have been made in insulin purity
and availability with the development of recombinant DNA
technology. Various formulations with different time-actions
have also been developed. Currently, there are generally
seven commercially available insulin formulations: Regular
insulin, semilente insulin, globin insulin, isophane insulin,
insulin zinc suspension, protamine zinc insulin, and
Ultralente insulin.
Despite the array of formulations available, subcutaneous
injection therapy still falls short of providing a patient
with convenient regulation and normalized glycemic control.
Frequent excursions from normal glycemia levels over a
patient's lifetime lead to hyper-or hypoglycemia, and long
term complications including retinopathy, neuropathy,
nephropathy, and micro- and macroangiopathy.
To help avoid extreme glycemic levels, diabetics often
practice multiple injection therapy whereby insulin is
administered with each meal. However, this therapy has not
yet been optimized. The most rapid-acting insulin
commercially available peaks too late after injection and
lasts too long to optimally control glucose levels.
Therefore, considerable effort has been devoted to create
2

insulin formulations and insulin analog formulations that
alter the kinetics of the subcutaneous absorption process.
Because all commercial pharmaceutical formulations of insulin
contain insulin in the self-associated state and
predominately in the hexamer form, it is believed that the
rate-limiting step for the absorption of insulin from the
subcutaneous injection depot to the bloodstream is the
dissociation of the self-aggregated insulin hexamer.
Recently, monomeric insulin analogs have been developed that
are less prone to association to higher molecular weight
forms than human insulin. This lack of self-association is
due to modifications in the amino acid sequence of human
insulin that decrease association by primarily disrupting the
formation of dimers. See, e.g., Brems et al. , Protein
Engineering, 55:6, 527-533 (1992) and Brange et al. , Nature,
333:679-682 (1988) . Accordingly, monomeric insulin analogs
possess a comparatively more rapid onset of activity while
retaining the biological activity of native human insulin.
These insulin analogs provide a rapid absorption to place
injection time and peak action of insulin into closer
proximity with postprandial glucose excursion associated in
the response to a meal.
The physical properties and characteristics of monomeric
analogs are not analogous to insulin. For example, Brems et
al. disclose that various monomeric analogs have little, or
no, Zn-induced association. Any association that is observed
is to a multitude of higher molecular weight forms. This
differs dramatically from insulin, which is almost
exclusively in an ordered, hexamer conformation in the
presence of zinc. Brange et al. Diabetes Care 13 : 923-954
(1990) . The lack of association attributes to the fast
acting characteristics of the analogs. Because the analogs
have lower tendency to associate, it is quite surprising that
a monomeric insulin analog can be formulated to provide an
intermediate duration of action.
3

The present invention provides a monomeric insulin analog
formulation that yields upon use an intermediate duration of
action. The invention further provides a novel protamine
crystal called insulin analog-NPD. The present invention
also provides a mixture of insulin analog-NPD and soluble
monomeric insulin analog. This mixture provides a rapid
onset of action and an intermediate duration of action.
Accordingly, the mixture possesses advantages over both
insulin and the monomeric analog. The present invention
further provides for a process for preparing uniform crystals
of insulin analog-NPD.
SUMMARY OF THE INVENTION
This invention provides an insulin analog-protamine
formulation, which comprises: a monomeric insulin analog,
protamine, zinc, and a phenolic derivative.
The invention further provides a crystalline insulin analog-
protamine complex. This complex has been defined as insulin
analog-NPD. LysB28ProB29-human insulin-NPD comprises: a
LysB28ProB29-human insulin, about 0.27 to about 0.32 mg
protamine/100 U of insulin analog, about 0.35 to about 0.9 %
zinc by weight, and a phenolic derivative.
This invention additionally provides a process for preparing
LysB28ProB29-human insulin-NPD, which comprises:
combining an aqueous solution of LysB28ProB29-human insulin
in a hexamer association state, and a protamine solution at a
temperature from about 8° to about 22°C;
said aqueous solution comprising from about 0.35 to about
0.9% zinc by weight, LysB28ProB29-human insulin, and a
phenolic derivative at a pH of about 7.1 to about 7.6;
said protamine solution comprising protamine at a pH of about
7.1 to about 7.6 such that the final concentration of
protamine is about 0.27 to about 0.32 mg protamine/100 U of
insulin analog.
4

The invention also provides formulations that are both rapid
and intermediate acting. The formulations are mixtures of
monomeric insulin analog and crystalline insulin analog-NPD,
wherein the ratio by weight of the two components is about 1-
99:99-1.
Finally, the invention provides a method of treating a
patient suffering from diabetes mellitus, which comprises
administering to said patient a pharmaceutical composition
containing insulin analog-protamine crystals.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a graphical representation of the profile of
action of LysB28ProB29-hI-NPD and human insulin-NPH. The
graph is mU/ml versus the Time of Infusion. The figure
demonstrates the advantages of the present invention.
FIGURE 2 presents a picture of AspB28-human insulin-protamine
crystals of the present invention. The picture was taken at
1000X magnification with differential contrast.
FIGURE 3 presents a picture of LysB28ProB29-human insulin-
protamine crystals of the present invention. The picture was
taken at 1000X magnification with differential contrast.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the invention provides various formulations
of a monomeric insulin analog. The term "monomeric insulin
analog" or "insulin analog" as used herein is a fast-acting
insulin analog that is less prone to dimerization or self-
association. Monomeric insulin analog is human insulin
wherein Pro at postion B28 is substituted with Asp, Lys, Leu,
Val, or Ala, and Lys at position B29 is Lysine or Proline;
des(B28-B30) ; or des(B27). Monomeric insulin analogs are
described in Chance et al. , EPO publication number 383 472,
5

and Brange et al. , EPO publication 214 826, and are herein
incorporated by reference.
One skilled in the art would recognize that other
modifications to the monomeric insulin analog are possible.
These modifications are widely accepted in the art and
include replacement of the histidine residue at position BIO
with aspartic acid; replacement of the phenylalanine residue
at position Bl with aspartic acid; replacement of the
threonine residue at position B30 with alanine; replacement
of the serine residue at position B9 with aspartic acid;
deletion of amino acids at position Bl alone or in
combination with a deletion at position B2; and deletion of
threonine from position B30.
All amino acid abbreviations used in this disclosure are
those accepted by the United States Patent & Trademark Office
as set forth in 37 C.F.R. § 1.822(b)(2). Particularly
preferred monomeric insulin analogs are LysB28ProB29-human
insulin (B28 is Lys; B29 is Pro) and AspB28-human insulin
(B28 is Asp).
The term "monomeric insulin analog-NPD" or "insulin analog-
NPD" is a suspension of crystalline insulin analog and
protamine in a formulation. NPD is Neutral Protamine
formulation according to DeFelippis. The composition is
prepared in accordance to the claimed process described
herein. A related term "insulin analog NPD crystals,"
"crystalline insulin analog-NPD," or "LysB28proB29-human
insulin-protamine crystals" refer to the insulin analog-
protamine crystals in the NPD formulation.
The term "treating," as used herein, describes the management
and care of a patient for the purpose of combating the
disease, condition, or disorder and includes the
administration of a compound of present invention to prevent
the onset of the symptoms or complications, alleviating the
6

symptoms or complications, or eliminating the disease,
condition, or disorder.
The term "isotonicity agent" refers to an agent that is
physiologically tolerated and embarks a suitable tonicity to
the formulation to prevent the net flow of water across the
cell membrane. Compounds, such as glycerin, are commonly
used for such purposes at known concentrations. The
concentration of the isotonicity agent is in the range known
in the art for insulin formulations.
The term "phenolic derivative" is m-cresol, phenol or
preferably a mixture of m-cresol and phenol.
The term "free base basis" indicates the amount of protamine
in the formulation. Free base basis corrects for the water
and salt content of the protamine salts commercially
available and commonly used in parenteral formulations. The
preferred protamine, protamine sulfate, is approximately 80%
protamine.
The term "IU" or "U" is international unit.
The term "isophane ratio" is the equilibrium amount of
protamine necessary to complex with the analog as taught by
Krayenbuhl and Rosenberg, STENO MEMORIAL HOSPITAL REPORT
(COPENHAGEN), 1:60 (1946). The isophane ratio is determined by
titration in a manner well known in the art and described in
Krayenbuhl, et al.
The present invention provides an insulin analog-protamine
formulation, which comprises: a monomeric insulin analog,
protamine, zinc, and a phenolic derivative. The
concentration of protamine is preferably about 0.2 to about
1.5 mg of protamine to 100 U of insulin analog on a free base
basis. Most preferably, the range of protamine is about 0.27
mg/100 U to about 0.35 mg/100 U. The concentration of zinc
7

is from about 0.35 to about 0.9 % on a weight basis.
Preferably, the concentration of zinc is about 0.7 %.
The phenolic derivative is m-cresol, phenol or a mixture of
m-cresol and phenol. Preferably the phenolic derivative is
m-cresol and phenol. The concentration of the phenolic
derivative is known to one skilled in the art. The
concentrations must be sufficient to maintain preservative
effectiveness, i.e., retard microbial growth. In general,
the concentration of phenolic is, for example in the range of
1.0 mg/mL to 6.0 mg/mL; preferably greater than about
2.5 mg/mL. The most preferred concentration is about
3 mg/mL. The presence of a phenolic derivative is critical
because it acts to complex the analog, protamine and zinc in
addition to serving as a preservative. However, it is
believed that only one molecule of phenol per molecule of
insulin analog is bound to the crystal structure.
Preferably, an isotonicity agent is added to the formulation.
The preferred isotonicity agent is glycerin. The
concentration of the isotonicity agent is, for example 14
mg/mL to 18 mg/mL, preferably about 16 mg/mL.
The pH of the formulation may be buffered with a
physiologically tolerated buffer, preferably a phosphate
buffer, like dibasic sodium phosphate. Other physiologically
tolerated buffers include TRIS, sodium acetate, or sodium
citrate. The selection and concentration of buffer is known
in the art. Preferably, the concentration is, for example,
about 1.5 mg/mL to 5.0 mg/mL; preferably 3.8 mg/mL.
The present invention further provides specific conditions
under which the insulin analog-protamine exists as a stable
crystal. Formulations of these crystals are defined as
insulin analog-NPD. Insulin analog-NPD is a formulated
suspension of insulin analog-NPD crystals and yields upon use
an intermediate duration of action. The profile of activity
8

of insulin analog-NPD is quite surprising in view of the lack
of self-association of the monomeric analog.
The ability to form an intermediate acting formulation with a
monomeric analog is demonstrated in FIGURE I. FIGURE I
discloses a profile of action for LysB28ProB29-hI-NPD and
human insulin-NPH. The NPD profile is similar to insulin-
NPH. The duration of action for the NPD formulation and the
insulin-NPH formulation are approximately equal. However,
most significantly, the present formulation rises more
rapidly and remains stable for a longer period than insulin-
NPH. This difference is quite unexpected in view of the
fast-acting profile of the monomeric analog.
A particularly preferred insulin analog-protamine
formulation, LysB28ProB29-human insulin-NPD, comprises:
LysB28ProB29-human insulin, about 0.27 to about 0.32 mg
protamine/100 U of insulin analog, about 0.35 to about 0.9 %
zinc by weight, and a phenolic derivative. The concentration
of protamine is preferably 0.3 mg/100 U on a free base basis.
The invention also provides the process for preparing
LysB28ProB29-human insulin-protamine crystals, which
comprises:
combining an aqueous solution of LysB28ProB29-human insulin
in a hexamer association state, and a protamine solution at a
temperature from about 8° to about 22°C;
said aqueous solution comprising from about 0.35 to about
0.9% zinc by weight, LysB28ProB29-human insulin, and a
phenolic derivative at a pH of about 7.1 to about 7.6;
said protamine solution comprising protamine at a pH of about
7.1 to about 7.6 such that the final concentration of
protamine is about 0.27 to about 0.32 mg protamine/100 U of
insulin analog.
At the time of invention it was known that monomeric insulin
analogs have a lesser tendency to associate and form
hexamers. The conditions necessary to cause the monomeric
9

insulin analogs to associate with protamine to form crystals
were previously unknown in the art. Previous studies relate
to insulin. The teachings regarding the preparation of
insulin-NPH (neutral protamine formulation according to
Hagedorn) or isophane insulin formulations by Krayenbiihl and
Rosenberg, STENO MEMORIAL HOSPITAL REPORT (COPENHAGEN), 1:60 (1946)
are not relevant in view of the distinct properties of the
monomeric insulin analogs. In fact, the commercial process
of producing Humulin-N™ (insulin-NPH), an acid-neutral
process, does not produce crystalline insulin analog-NPD.
Most significantly, it has been found that the parameters in
the present process -- namely, the temperature of the
crystallization and the formation of a hexamer complex of the
insulin analog, zinc, and the phenolic derivative are
critical limitations to the formation of stable,
LysB28ProB29-hI-NPD crystals.
The temperature of the crystallization must be from about 8°C
to about 22°C, preferably from 13°C to 17°C. If the
temperature is outside of this range, a largely amorphous
insulin analog-protamine formulation results.
It is also critical that the insulin analog be transformed to
a hexamer state prior to the crystallization. The
crystallization results in an amorphous product when the
process is carried out with a monomeric association state.
Crystals form without agitation in five to thirty-six hours
hours. Good quality crystals are generally formed in 24
hours.
Soluble monomeric insulin analog is complexed to a hexamer
association state by suspending solid monomeric analog in a
diluent containing the phenolic derivative and adding zinc
until the concentration is from about 0.35 % to about 0.9 %
on a weight basis. Zinc is preferably added as a salt.
Representative examples of zinc salts include zinc acetate,
zinc bromide, zinc chloride, zinc fluoride, zinc iodide and
10

zinc sulfate. The skilled artisan will recognize that there
are many other zinc salts that also might be used in the
process of the present invention. Preferably, zinc acetate
or zinc chloride is used.
Dissolution of the insulin analog in the diluent may be aided
by what is commonly known as an acid dissolution. In an acid
dissolution, the pH is lowered to about 3.0 to 3.5 with a
physiologically tolerated acid, preferably HC1, to increase
solubility of the analog. Other physiologically tolerated
acids include acetic acid, citric acid, and phosphoric acid.
The pH is then adjusted with a physiologically tolerated
base, preferably NaOH to about 7.1 to 7.6 for the
crystallization. Other physiologically tolerated bases
include KOH and ammonium hydroxide.
Most significantly, the process of producing LysB28proB29_hI_
NPD complex is sensitive to the concentration of NaCl. If
the concentration exceeds about 4 mg/mL, the insulin analog-
NPD crystals become mixed with amorphous product.
Accordingly, it is preferred that the monomeric analog is
dissolved at neutral pH to avoid the formation of salt ions.
Alternatively, the analog may be dissolved in the diluent at
an acid pH prior to the addition of the buffer. This reduces
the concentration of salts generated due to the pH
adjustment. However, the order that the constituents are
added is not critical to the formation of the hexamer or the
amorphous formulation.
As previously disclosed, an isotonicity agent may be added to
the formulations of the present invention. The addition of
the isotonicity agent can be to the analog solution, to the
protamine solution, or to the final insulin analog-NPD
formulation. Likewise, the addition of the physiologically
tolerated buffer may be added to the analog solution, to the
protamine solution, or to the final insulin analog-NPD
formulation. However, it is preferred that both the analog
solution and the protamine solution contain the isotonicity
11

agent and the buffer prior to combining the aqueous solution
and the protamine. Because of the NaCl effects on the
process for producing crystalline insulin-analog-NPD,
glycerin is the preferred isotonicity agent.
The invention also provides insulin analog formulations,
which comprise mixtures of insulin analog-NPD as a
crystalline solid and soluble insulin analog. These mixtures
are prepared in a range of about 1:99 to 99:1, by volume
suspended insulin analog-NPD to soluble insulin analog. The
soluble insulin analog is a monomeric insulin analog
dissolved in an aqueous diluent comprising: zinc, a phenolic
derivative, an isotonicity agent, and buffer. The
concentrations described in the diluent are the same as
previously disclosed herein. Preferably the ratio of insulin
analog-NPD to soluble insulin analog is 25:75 to 75:25; and
more preferably, 50:50. The mixtures are readily prepared by
mixing the individual constituents.
The mixed formulations of the present invention are
especially suitable for the treatment of diabetes mellitus
because of the combination of a rapid onset of action and
prolonged duration. These mixtures allow "fine control" by
varying the amount of each individual constituent based on
the needs, diet, and physical activity of the patient. The
mixture of suspended insulin-analog-NPD and soluble insulin
analog are also advantageous because they are homogeneous,
i.e., any equilibrium exchange between the suspended crystals
and soluble insulin analog is transparent.
The insulin analogs of the present invention can be prepared
by any of a variety of recognized peptide synthesis
techniques including classical (solution) methods, solid
phase methods, semi synthetic methods, and more recent
recombinant DNA methods. For example, Chance et al., EPO
publication number 383 472, and Brange et al. , EPO 214 826,
disclose the preparation of various monomeric analogs.
12

The following examples are provided merely to further
illustrate the preparation of the insulin analogs and the
invention. The scope of the invention is not construed as
merely consisting of the following examples.
EXAMPLE
Example 1: Preparation of LysB28ProB29-hI-NPD
A solution of LysB28ProB29-human insulin (LysB28ProB29-hI) at
200 IU/mL (U200) concentration was prepared by dissolving
zinc containing crystals of LysB28ProB29-hI in a
preservative/buffer system containing: 1.6 mg/mL m-cresol,
0.73 mg/mL phenol (equivalent to 0.65 mg/mL phenol calculated
as 89 %) , 16 mg/mL glycerin, and 3.78 mg/mL of dibasic sodium
phosphate buffer. The endogenous zinc level in the crystals
was supplemented by adding an appropriate volume of an acidic
ZnO solution (10 mg/mL) to achieve a final concentration of
0.025 mg/100 IU (0.7%). Dissolution of LysB28ProB29-hI was
accomplished at ambient temperature by lowering the pH to
about 3 with /mL volumes of 5 M HC1. After the solution had
clarified, the pH was readjusted to 7.5 with /mL volumes of 5
M NaOH.
A protamine solution was prepared by dissolving enough solid
protamine sulfate in the preservative/buffer solution to
achieve a final concentration of 0.6 mg/100 IU calculated on
a free base basis. The pH of this solution was adjusted to
7.5 and equilibrated at 15°C.
Both solutions were diluted to final concentration with water
for injection and filtered. 5 mL aliquots of the
LysB28ProB29-hI subsection were filled into separate clean
glass vials, and the samples were incubated in a water bath
at 15 °C. After appropriate time for equilibration (15
minutes), precipitation was induced by rapidly adding 5 mL of
the protamine solution to the LysB28ProB29-hI samples. The
13

crystallization was allowed to proceed about 24 hours at
15°C.
Example 2: Preparation of LysB28proB29-hI-NPD
The process is identical to Example 1, except that the
dissolution of LysB28ProB29-hI occurs at neutral pH. The
process was carried out such that the final pH was 7.4.
Example 3: Preparation of LysB28proB29_hI_NPD
Insulin analog-NPD was prepared in a manner analogous to
Example 1, but the acid dissolution of LysB28ProB29-hI was
carried out in the presence of all excipients except the
dibasic sodium phosphate buffer. Solid dibasic sodium
phosphate is added after the insulin analog solution was
returned to pH 7.4. The addition of dibasic sodium
phosphate clarified the solution.
Example 4: Preparation of insulin analog-NPD mixture
formulations
Mixtures of intermediate and rapid acting LysB28ProB29-hI
formulations are prepared as follows. The intermediate
acting, suspension preparation is prepared by the methods
described in Example 3 and serves as the intermediate acting
section for the mixture. A separate solution of
LySB28proB29_hI (100o IU) is prepared by dissolving zinc-
containing LysB28ProB29-hI crystals at ambient temperature in
the diluent described in Example 1. The endogenous zinc
level of LysB28ProB29-hI in this solution is supplemented by
the addition of acidic ZnO solution to match the level in the
suspension section (i.e., 0.025 mg/100 IU (0.7%)). Water for
injection is used to dilute the solution to final
concentration after the pH is adjusted to 7.4 using 10%
solutions of HC1 and/or NaOH. This solution is the rapid
acting section of the mixtures. The final mixture is
prepared by combining appropriate volumes of the intermediate
14

and rapid acting subsections to achieve the desired ratio. A
50/50 mixture is prepared by combining 1 part of the
intermediate acting section with 1 part of the rapid acting
section by volume.
Example 5: Effect of ionic strength on LysB28ProB29-hI
protamine crystallization
The effect of ionic strength on the crystallization was
evaluated by the addition of NaCl to the LysB28ProB29-hI
section prior to mixing with protamine. NaCl was added so
that the total concentration was 20, 30, and 40 mM (1.2, 1.8,
and 2.3 mg/ml). The volume particle size displayed multi-
modal behaviour (additional peaks at small particle sizes) ,
as the NaCl concentration was increased. The volume mean
particle size decreased as NaCl concentration was increased
indicating an increase in amorphous material. Results of
particle size vs. NaCl concentration are as follows:

[NaCl] Volume Mean Particle Size
(mm)
13 mM 3.9
20 mM 3.5
30 mM 3.3
40 mM 3.2
The microscope analysis showed that all samples contained a
mixture of amorphous and crystalline material. The sample
containing 40 mM NaCl had mostly amorphous material and very
few crystals.
Example 6: Comparative dynamics of LysB28proB29-hI-NPD and
human insulin-NPH
This study was carried out in a conscious dog model. Prior
to the commencement of the study, three basal samples were
taken. An infusion of somatostatin (0.3 mg/Kg-min.) was
initiated. After a 10 minute interval, a subcutaneous
15

injection of either NPD or NPH was administered. Frequent
monitoring of plasma glucose was initiated and a variable
glucose (20%) infusion was given so as to maintain near-
normal glycemia. Samples were taken throughout and were
analyzed for immunoreactive insulin (Linco antibody) and
glucose. The results are illustrated in FIGURE 1.
Example 7: Preparation of Asp(B28) Analog-Protamine Crystals
A subsection of Asp(B28)-hI at 200 IU/mL (U200) concentration
was prepared by dissolving lyophilized bulk (95% purity) in a
preservative/buffer system containing: 1.6 mg/mL m-cresol,
0.73 mg/mL phenol (equivalent to 0.65 mg/mL phenol calculated
as 89 %), 16 mg/mL glycerin, and 3.78 mg/mL dibasic sodium
phosphate. Zinc was added to the system using an appropriate
volume of an acidic ZnO solution (10 mg/mL) to obtain a final
concentration of 0.025 mg/100 IU. Dissolution of Asp(B28)
was achieved at ambient temperature at neutral pH. The final
pH of the section was 7.4.
A crystallization was carried as described in Example 2.
Final protamine concentrations of 0.3 mg/lOOU, 0.35 mg/lOOU,
and 0.4 mg/lOOU were investigated. These protamine
concentration correspond to 2.9%, 9.3% and 10.5% respectively
on a weight/weight basis. Incubation temperatures included 5
°C (0.3 mg/lOOU only), 15 °C and 22 °C. After 24 hr. at
these temperatures, samples were analyzed for crystal
formation. Results as determined by microscopy illustrate a
mixture of a few crystals and amorphous product.
Example 8: Preparation of Asp(B28) Analog-Protamine Crystals
The crystallization Asp(B28) Protamine was performed as
described in Example 7, except that the protein was first
dissolved in a buffer-free diluent. The addition of the
acidic ZnO stock was sufficient to acidify the sample to pH
2.0-2.5. After the solution had clarified, the pH was
readjusted to approximately pH 7 with mL volumes of 5 N NaOH.
16

Sodium phosphate, dibasic, was added using a concentrated
stock solution at 47.25 mg/mL to achieve the final
concentration of 3.78 mg/mL. The subsection was adjusted to
pH 7.4 using mL quantities of HC1.
Crystallization was initiated by combining the Asp(B28) and
protamine sections, as described in previous examples. Final
protamine concentrations of 0.3 mg/lOOU, 0.35 mg/lOOU, and
0.4 mg/lOOU were investigated. Incubation temperatures
included 15 °C and 22 °C. After 24 hr. at these
temperatures, samples were analyzed for crystal formation.
Results as determined by microscopy illustrate a mixture of a
crystals and amorphous material.
Example 9: Preparation of Leu(B28)Pro(B29) Analog-Protamine
Crystals
A subsection of Leu(B28)Pro(B29) (93% purity) at 200 IU/mL
(U200) concentration was prepared as described in Example 8
using an acid dissolution of the bulk followed by pH
adjustment with 5N NaOH to pH 7.4. Crystallization was as
described above. Final protamine concentrations of 0.3
mg/lOOU, 0.35 mg/lOOU, and 0.4 mg/lOOU were investigated.
Incubation temperatures included 5 °C, 15 °C and 22 °C.
After 24 hr. at these temperatures, all samples contain some
crystals, but were primarily amorphous as determined by
microscopy.
Example 10: Des(B27)hl-protamine crystals
A subsection of DesThr(B27) (97.37 % purity) at 200 IU/mL
(U200) concentration was prepared as described in Example 8
using an acid dissolution of the bulk followed by pH
adjustment with 5N NaOH to pH 7.4. A crystallization was
carried out as described in Example 8. Final protamine
concentrations of 0.3 mg/lOOU, 0.35 mg/lOOU, and 0.4 mg/lOOU
were investigated. Incubation temperatures included 15 °C
and 22 °C. After 24 hr. at these temperatures, all samples
17

were primarily amorphous as determined by microscopy.
Qualitatively, crystals were observed.
Example 11: Des(B28-B30)hl-protamine
A subsection of Des(28-30) (96.3 % purity) at 200 IU/mL
(U200) concentration was prepared as described in Example 8
using an acid dissolution of the bulk followed by pH
adjustment with 5N NaOH to pH 7.4. A crystallization was
attempted using the neutral/neutral combination method of the
protein and protamine sections as described above. Final
protamine concentrations of 0.3 mg/lOOU, 0.35 mg/lOOU, and
0.4 mg/lOOU were investigated. Incubation temperatures
included 15 °C and 22 °C. After 24 hr. at these
temperatures, all samples were primarily amorphous as
determined by microscopy. Qualitatively, crystals were
observed. The crystals were well defined.
Example 12: Asp(B28) Analog-Protamine
A insulin Asp(B28)-human insulin analog solution was prepared
by dissolving 16.6 mg of the protein in 1 mL of a solution
containing 3.2 mg/mL m-cresol, 1.3 mg/mL phenol and 32 mg/mL
glycerin. A 14.4 mL aliquot of an acidic zinc stock solution
(10 mg/mL in Zn2+ , prepared by dissolving 0.311 g of zinc
oxide in 5 mL of 10% HC1 and diluting to 25 mL with water) .
The solution pH was 2.3 which allowed for complete
dissolution of the protein. A 10 mL aliquot of 10% NaOH was
added to adjust the pH to 7.06. To the solution was added
100 mL of 0.2 8 M dibasic sodium phosphate, pH 7.0 which
increased the solution pH to 7.27. A 870 mL aliquot of water
for injection was added to the solution. Additional 10% HC1
(1 mL) and NaOH (0.7 mL) were added, and the final volume of
the solution was brought to 2 mL with water for injection
resulting in a final pH of 7.26. The solution was filtered
through a 0.2 mm Supor" Acrodisc" 13, Gelman Sciences) filter
before use.
18

Protamine stock solutions were prepared by dissolving
protamine sulfate in a solution containing 1.6 mg/mL m-
cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM
dibasic sodium phosphate. The final pH of the solution was
adjusted to 7.3. The final protamine concentration was 0.60
mg/lOOU on a free base basis. Both solutions were filtered
through 0.22 |um (Millipore Sterivex™-GV) filter units before
use.
Crystallization was achieved by mixing the Asp(B28)-human
insulin solution in a 1:1 ratio at controlled temperature as
outlined in Table 1. The final mixture conditions were 3.94
mg/mL Asp(B28)-human insulin, 0.0359 mg/mL (0.9%) zinc ions,
1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin, 14
mM dibasic sodium phosphate and 0.30 mg/lOOU of protamine at
pH 7.3. Specifically, 50-200 mL portions of the AspB28-human
insulin solution were transferred to glass vials, and the
samples were equilibrated to 4, 8, 15 or 23 (ambient
temperature) °C. Portions of both protamine solutions were
also equilibrated at these temperatures. After 15-20
minutes, an equivalent volume of either protamine solution
was pipetted into the Asp(B28)-human insulin samples. The
mixture was gently swirled, capped and then left quiescent at
controlled temperature during the crystallization period.
All of the samples were examined by microscopy after 24 hours
and found to be predominantly amorphous. After 48 hours, the
sample containing 0.30 mg/100 U of protamine and incubated at
15 °C showed extensive amounts of needle-like crystals and
some amorphous material.
Example 13:
A insulin Asp(B28)-human insulin analog solution was prepared
by dissolving 10.62 mg of the protein in 0.71 mL of a
solution containing 3.2 mg/mL m-cresol, 1.3 mg/mL phenol and
32 mg/mL glycerin. A 10.2 mL aliquot of an acidic zinc stock
solution (10 mg/mL in Zn2+, prepared by dissolving 0.311 g of
zinc oxide in 5 mL of 10% HC1 and diluting to 25 mL with
19

water). The solution pH was 2.3 which allowed for complete
dissolution of the protein. A 6.5 mL aliquot of 10% NaOH was
added to adjust the pH to 7.00. To the solution was added 71
H.L of 0.28 M dibasic sodium phosphate, pH 7.0 which increased
the solution pH to 7.26. A 620 mL aliquot of water for
injection was added to the solution. Additional 10% HCl (0.2
mL) and NaOH (0.6 )j.L) were added, and the final volume of the
solution was brought to 1.42 mL with water for injection
resulting in a final pH of 7.42. The solution was filtered
through a 0.2 mm Supor Acrodisc 13, Gelman Sciences) filter
before use.
A protamine stock solution was prepared by dissolving
protamine sulfate in a solution containing 1.6 mg/mL m-
cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM
dibasic sodium phosphate. The final pH of the solution was
adjusted to 7.4, and the final protamine concentration was
0.60 mg/lOOU on a free base basis. The solution was filtered
through a 0.22 urn (Millipore Sterivex™-GV) filter unit before
use.
Crystallization was achieved by mixing the Asp(B28)-human
insulin solution in a 1:1 ratio with the protamine solution
as described in Example 12 at controlled temperatures 13°C,
15°C, 17°C and 23°C. The results are presented in Table 1.
The final mixture conditions were 3.74 mg/mL Asp(B28)-human
insulin, 0.0359 mg/mL (0.9%) zinc ions, 1.6 mg/mL m-cresol,
0.65 mg/mL phenol, 16 mg/mL glycerin, 14 mM dibasic sodium
phosphate and 0.3 0 mg/lOOU of protamine at pH 7.4. Four
different crystallization temperatures were evaluated. A 1
mL aliquot of the AspB28-human insulin equilibrated at 15 °C
was mixed with 1 mL of the protamine solution adjusted to the
same temperature. After gentle swirling the preparation was
left quiescent at 15 °C. Another sample was prepared by
equilibrating 100 mL of the Asp (B28)-human insulin solution
to 13 °C, and then combining with 100 mL of the protamine
solution adjusted to the same temperature. The final mixture
was incubated at 13 °C. The third sample was prepared in a
20

similar manner except that the two 100 mL aliquots were
equilibrated, combined and then incubated at 17 °C. The
final solution was prepared by mixing ambient temperature
equilibrated, 80 mL aliquots of the Asp (B28)-human insulin
and protamine solutions and incubating at ambient temperature
(23°C). All samples were evaluated by microscopy after 24
hours and other time intervals thereafter as listed in Table
1.
Table 1
Crystallization Conditionsa and Results from Microscopy

a All solutions also contained 0.9% zinc ions, 1.6 mg/mL m-
cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM
dibasic sodium phosphate, pH 7.4.
21

b Crystallization outcome was evaluated by microscopy at 600X
(Nikon Optiphot 66 microscope) or 100OX (Zeiss Axioplan
microscope with differential interface contrast)
magnification. Both microscopes were equipped with
accessories for photography.
Crystals prepared in accordance with the above examples are
illustrated in Figure 2 and Figure 3.
22

WE CLAIM:

1. A parenteral pharmaceutical LysB28ProB29 -human
insulin-protamine formulation, which comprises: LysB28ProB29-
human insulin; about 0.27 to about 0.32 mg protamine/100 IU
of insulin analog, about 0.35 to about 0.9 % zinc by weight,
and about 1.0 to about 6.0 mg/mL phenolic derivative.
2. A formulation of Claim 1, which comprises about 0.2
to about 1.5. mg protamine/100 IU insulin analog; about 0.35
to about 0.9% zinc by weight; and about 1.0 to about 6.0
mg/mL phenolic derivative.
3. A parenteral pharmaceutical formulation of Claim 2,
which comprises: LysB28ProB29-human insulin, about 0.27 to
about 0.32 mg protamine/100 IU insulin analog, and about 0.35
to about 0.9% zinc by weight.
4. A parenteral pharmaceutical formulation, which
comprises: LysB28ProB29-human insulin, about 0.3 mg
protamine/100 IU of insulin analog, about 0.7% zinc by
weight, about 1.7 mg/mL m-cresol, about 0.7 mg/mL phenol,
about 16 mg/mL glycerin and about 3.7 8 mg/mL dibasic sodium
phosphate.
5. A parenteral pharmaceutical formulation as claimed
in any of claims 1 through 4, which comprises: LysB28ProB29-
human insulin; about 0.27 to about 0.32 mg protamine/100 IU
of insulin analog, about 0.35 to about 0.9 % zinc by weight,
and about 1.0 to about 6.0 mg/mL phenolic derivative and
optionally comprises soluble LysB28ProB29-human insulin analog.
6. A parenteral pharmaceutical formulation, which
comprises: a mixture of soluble LysB28ProB29-human insulin and
Lys Pro -human insulin-protamine crystals; wherein the
ratio by weight of the two components is about 1:99 to 99:1
23

LysB28ProB29-human insulin to LysB28ProB29-human insulin-
protamine crystals.
7. A parenteral pharmaceutical formulation of Claim 6,
wherein the ratio by weight of the two components is about
75:25 to 25:75.
8. A parenteral pharmaceutical formulation of Claim 7,
wherein the ratio by weight of the two components is 50:50,
75:25, or 25:75.
9. A process for preparing the complex in claims 1 or
claim 2, which comprises: combining LysB28ProB29-human insulin,
protamine, zinc and a phenolic derivative in an aqueous
solvent and allowing the complex to form.
10. A process for preparing LysB28ProB29-human insulin-
protamine crystals, which comprises: combining an aqueous
solution of LysB28ProB29-human insulin in a hexamer association
state, and a protamine solution at a temperature from about
8° to about 22°C; said aqueous solution comprising from about
0.35 to about 0.9% zinc by weight, LysB28ProB29-human insulin,
and a phenolic derivative at a pH of about 7.1 to about 7.6;
said protamine solution comprising protamine at a pH of about
7.1 to about 7.6 such that the final concentration of
protamine is about 0.27 to about 0.32 mg protamine/100 IU of
insulin analog.
11. The process of Claim 10, wherein the temperature is
15°C; the zinc concentration is 0.7% to 0.9%; and the
protamine concentration is 0.3 mg/100 IU of insulin analog.
12. A process of preparing a parenteral pharmaceutical
formulation as claimed in any of claims 1 through 9, which
comprises: suspending LysB28ProB29 -human insulin-protamine
crystals in a pharmaceutically acceptable diluent.
24

25
13. Insulin analog-protamine crystals whenever prepared
by a process according to any one of Claims 9, 10, or 11.
14. A parenteral pharmaceutical LysB28ProB29-human
insulin-protamine formulation, a process for preparing the
complex, and a process for preparing LysB28ProB29-human
insulin-protamine crystals, substantially as hereindescribed
with reference to the accompanying examples and drawings.

The present invention discloses various parenteral
pharmaceutical formulations, which comprise: a monomeric
insulin analog, zinc, protamine, and phenolic derivative.
The analog formulations provide a prolonged duration of
action. A process for preparing insulin analog-protamine
formulations is also described.