SODIUM CHLORIDE SOLUTION FOR DRUG RECONSTITUTION OR DILUTION
[0001] This application claims priority to U S Serial No 60/732,221, filed November
1, 2005, which is hereby incorporated by reference in its entirety
[0002] All patents, patent applications and publications cited herein are hereby
incorporated by reference in therr entirety The disclosures of these publications in therr
entireties are hereby incorporated by reference into this application in order to more fully
describe the state of the art as known to those skilled therein as of the date of the invention
described and claimed herein
[0003] A portion of the disclosure of this patent document contains material that is
subject to copyright protection The copyright owner has no objection to the facsimile
reproduction by anyone of the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise reserves all copyright nghts
whatsoever
BACKGROUND OF THE INVENTION
[0004] When whole blood is mixed with a drug solution, such as in the intravenous
(TV) line during administration of an IV injectable drug, rouleaux or erythiocyte aggregation
(also called red blood cell agglutination) may occur if the drug solution does not contain
sufficient ionic strength Erythrocyte aggregation occurs, for example, with 5% dextrose in
water (a common large volume parenteral solution) and with many pharmaceutical products
that have low ionic strength
[0005] Pharmaceutical products are often lyophihzed, and therefore need to be
reconstituted with a solution prior to parenteral injection However, when lyophihzed
products are reconstituted with low ionic strength solutions, the resultant preparation can also
cause erythiocyte aggregation Although reconstitution of lyophihzed cakes with normal
saline solution (0 9% NaCl) as opposed to sterile water for injection (sWFI) would provide
additional ionic strength, the resulting drug solution may be hypertonic and could have
undesirable side effects upon injection
[0006] Thus, there remains a need for a solution that can be used to reconstitute
lyophihzed cakes or to dilute pharmaceutical solutions to provide a resultant preparation for
injection that is isotonic with respect to plasma and has sufficient ionic strength such that it
does not cause erythrocyte aggregation

SUMMARY OF THE INVENTION
[0007] The invention provides the discovery that erythrocyte agglutination is caused
by low wnic strength solutions that come into contact with blood Thus, when
pharmaceutical formulations are prepared for intravenous injection by reconstitution or
dilution in low ionic strength solutions such as 5% dextrose, 3% dextran, or sWTT, the
resultant preparations may have the requisite osmolality to be about isotonic with respect to
blood, but they often do not have an ionic strength sufficient to prevent agglutination On the
other hand, when pharmaceutical formulations are prepared for intravenous injection by
reconstitution of dilution with high ionic strength solutions such as saline (0 9% NaCl or 154
mM NaCl), the resultant preparation may have a sufficient ionic strength to prevent
agglutination, but it may possess an osmolanty that is hypertonic with respect to blood,
thereby causing dehydration of red blood cells (RBCs), venous inflammation, and/or possibly
thrombophlebitis if repeated injections are frequent or chronic
[0008] In one aspect, the invention provides a method for preparing a pharmaceutical
formulation for intravenous injection, the method comprising adding an about 25 mM to
about 150 mM sodium chloride solution to the pharmaceutical formulation thereby resulting
in a formulation prepared for intravenous injection, wherein the prepared formulation is about
isotonic with respect to plasma or is slightly hypotonic or slightly hypertonic with respect to
plasma, and wherein the prepared formulation has a sufficient ionic strength to prevent
erythrocyte agglutination (or erythrocyte aggregation)
[0009] The prepared formulation is about isotonic with respect to plasma, for
example, when it has an osmolanty that is from about 270 mOsm/L to about 330 mOsm/L
The prepared formulation is slightly hypotonic with respect to plasma, for example, when it
has an osmolanty that is from about 220 mOsm/L to about 270 mOsm/L The prepared
formulation is slightly hypertonic with respect to plasma, for example, when it has an
osmolanty that is from about 330 mOsm/L to about 600 mOsm/L
[0010] In one aspect, the prepared formulation has an ionic strength that is sufficient
to prevent erythrocyte agglutination when it has, for example, at least about 25 mEq/L of Na+
and CI" ions In another aspect, the prepared formulation has an ionic strength that is
sufficient to prevent erythrocyte agglutination when it has, for example, at least about 40
mEq/L of Na+ and CI" ions In another aspect, the prepared formulation has an ionic strength
that is sufficient to prevent erythrocyte agglutination when it has, for example, at least about

40'mEq/L of Na+ and Cl- ions and less than about 150 mEq/L of Na+ and CI" ions In another
aspect, the prepared formulation has an ionic strength that is sufficient to prevent erythrocyte
agglutination when it has, for example, an ionic strength as measured in conductivity that is
at least about 2 5 mS/cm In another aspect, the prepared formulation has an lonic strength
that is sufficient to prevent erythrocyte agglutination when it has, for example, an ionic
strength as measured in conductivity that is at least about 4 0 mS/cm
[0011] The pharmaceutical formulation to be prepared for injection can be, for
example, a non-liquid formulation or a solution formulation A non-liquid formulation can
therefore be reconstituted into solution by a sodium chloride solution that has a sodium
chloride concentration of about 25-150 mM, 25-100 mM, 25-80 mM, 25-40 mM, 25-35 mM,
25-30 mM, or about 40 mM A liquid or a solution formulation can therefore be diluted by a
sodium chloride solution that has a sodium chloride concentration of about 25-150 mM, 25-
100 mM, 25-80 mM, 25-40 mM, 25-35 mM, 25-30 mM, or about 40 mM A non-liquid
formulation can be, for example, a lyophilized formulation
[0012] In one aspect, the sodium chloride solution that is added comprises from about
40 mM to about 150 mM sodium chloride In one aspect, the sodium chloride solution that is
added consists essentially of from about 40 mM to about 150 mM sodium chloride In one
aspect, the sodium chloride solution that is added comprises about 40 mM sodium chloride
In one aspect, the sodium chloride solution that is added consists essentially of a 40 mM
sodium chloride solution In one aspect, the sodium chloride solution that is added consists
essentially of a solution that has a sodium chloride solution that is about 40 mM ±10 mM
sodium chloride
[0013] In one aspect, wherein prior to the addition of the sodium chloride solution,
the pharmaceutical formulation does not contain an appreciable amount of an lomzing salt
An appreciable amount of an ionizing salt can be, for example, an amount that is greater than
about 5 mM In another aspect, an appreciable amount of an lomzing salt can be, for
example, an amount that is greater than about 25 mM If the pharmaceutical formulation is a
lyophilized formulation, it does not contain an appreciable amount of an ionizing salt if it
does not contain more than, for example, 5 mM or 25 mM of an ionizing salt when the
lyophilized formulation is reconstituted in water
[0014] In one aspect, wherein prior to the addition of the sodium chloride solution,
the pharmaceutical formulation comprises histidine, glycine, sucrose, and polysorbate In

another aspect, wherein prior to the addition of the sodium chloride solution, the
pharmaceutical formulation comprises histidine, glycine, sucrose, polysorbate, and a
therapeutic protein Herein, a therapeutic protein can be, for example, a pi otein used for
treating clotting disorders or for hemostasis, including but not limited to Factor VII, Factor
VIII, Factor DC, Factor XIII, antibodies, lelated analogues thereof, and derivatives thereof In
another aspect, wherein pnor to the addition of the sodium chloride solution, the
pharmaceutical formulation comprises histidine, glycine, sucrose, polysorbate, and Factor DC
(including lecombinant Factor DC (rFDC)) As used herein, Factor DC can include modified
versions of Factor DC, including for example, PEGylated Factor DC, protein fusions
comprising Factor DC such as albumin-Factor DC or immunoglobulin (whole or domains
thereof)-Factor DC, and glycosylated Factor DC
[0015] In one aspect, wherein the phannaceutical formulation is a lyophihzed
formulation, pnor to the addition of the sodium chloride solution, the formulation, measured
as if it was reconstituted in water (in a volume that is the same as the fill volume, i e, the
volume of the formulation pnor to lyophilization), comprises (a) from about 5 mM to about
30 mM histidine, (b) from about 0 1M to about 0 3M glycine, (c) from about 0 5 to about 2
percent sucrose, and (d) from about 0 001 to about 0 05 percent polysorbate (or from about
0 005 to about 0 05 percent) In one aspect, the formulation can further comprise, measured
if it was reconstituted in water, (e) from about 0 1 mg/mL to about 100 mg/mL or more of a
therapeutic pro tern, or from about 10, 50, 100, 200, 300, 400, 500, 1000, 2000 IU/mL
(international units/mL) or more of a therapeutic protein In one aspect, the formulation can
further comprise, measured if it was reconstituted in water, (e) from about 0 1 mg/mL to
about 100 mg/mL or more of Factor DC, or from about 0 4 mg/mL to about 20 mg/mL of
Factor DC, or from about 10, 50,100,200, 300,400, 500, 1000,2000 IU/mL (international
units/mL) or more of Factor DC
[0016] In one aspect, the invention provides a method for preventing erythrocyte
agglutination caused from intravenous injection, the method comprising reconstituting or
diluting a pharmaceutical formulation with an about 25 mM to about 150 mM sodium
chlonde solution such that the reconstituted or diluted pharmaceutical formulation has an
ionic strength sufficient to prevent erythrocyte agglutination when the reconstituted or diluted
pharmaceutical formulation is administered to a subject by intravenous injection
[0017] In one aspect, the invention provides a method for prepanng a lyophihzed
pharmaceutical formulation for intravenous injection, the method comprising reconstituting

the lyophihzed pharmaceutical formulation with an about 25 mM to an about 150 raM
sodium chloride solution such that after reconstitution, the formulation has an ionic strength
sufficient to prevent erythrocyte agglutination and an osmolanty that is about isotonic (or
slightly hypertonic or slightly hypotonic)
[0018] In one aspect, the lyophihzed formulation is reconstituted with the sodium
chloride solution, wherein the volume of the sodium chloride solution used for reconstitution
is less than the volume of the formulation pre-lyophihzation (z e, fill volume) In this
manner, the invention piovides a method for reducing the volume of the formulation to be
injected
[0019] In one aspect, the lyophihzed formulation is reconstituted with the sodium
chloride solution, wherein the volume of the sodium chloride solution used for reconstitution
is greater than the volume of the formulation pre-lyophihzation (z e, fill volume) In this
manner, the invention provides a method for maintaining the isotonicity of the formulation to
be injected
[0020] For example, a lyophihzed formulation can be reconstituted with a volume of
sodium chloride solution that is greater than the volume of the formulation pre-lyophihzation,
such as reconstitution with 5 mL of sodium chloride where the formulation volume pre-
lyophihzation is 4 mL Foi example, a lyophilized 4 mL formulation reconstituted in 4 mL
water is an isotonic solution containing 10 mM histidine, 260 mM glycine, 1% sucrose,
0 005% polysorbate, which is about 300 mOsm/L But if the lyophilized formulation is
reconstituted with 4 mL of a 40 mM NaCl (80 mOsm/L) solution, then the resultant
formulation would have a slightly hypertonic solution (300 mOsm/L + 80 mOsm/L = 380
mOsm/L) But if the lyophihzed formulation is reconstituted with 5 mL of a 40 mM NaCl
solution, then the resulting solution is about 8 mM (8 rnOsm/L) histidine, 208 mM (208
mOsm/L) glycine, 0 8% (24 mOsm/L) sucrose, 0 004% (negligible osmolanty) polysorbate,
and 40 mM (80 mOsm/L) NaCl, which is about 320 mOsm/L Thus, by reconstituting a pre-
lyophiiizanon formulation that is about isotonic with a volume of sodium chloride solution
i
that is greater than the fill volume, the invention can provide a resulting solution that is still
about isotonic In other words, reconstituting a pre-lyophihzation formulation that is about
isotonic with a volume of sodium chloride solution that is less than or about the same as the
fill volume can result in a solution that is slightly hypertonic To avoid this, the invention
provides a method for maintaining isotonicity by reconstituting a lyophihzed formulation

with a sodium chloride solution in a volume that is greater than the volume of the formulation
pre-lyophilization
[0021] In one aspect, the mventioi. provides a method for maintaining isotonicity of a
lyophilized formulation after reconstituucp, the method comprising reconstituting a
lyophilized formulation in a volume that is at least 20% greater than the volume of the
formulation pre-lyophilization, wherein tl ie formulation pre-lyophilization is about isotonic,
such that the reconstituted formulation is about isotonic and has a sufficient lonic strength to
prevent erythrocyte agglutination By reconstituting a lyophilized formulation in a volume
greater than its pre-lyophilization volume, the contribution to osmolality of the lyophilized
cake is decreased in direct pioportion to the increase of volume from reconstitution as
compared to pre-lyophihzation For example, a pre-lyophilization formulation with a tonicity
of 300 mOsm/L in a volume X, if reconstituted in a solution with volume Y that is 20%
greater than volume X, the 300 rnOsm/L is then 240 mOsm/L in volume Y (a 20% decrease in osmolality due to a 20% increase in volume) If the solution of volume Y is a sodium
chloride solution, then the contribution towards tonicity of the sodium chloride solution is
twice the concentration of sodium chloride in the solution For example, if the solution of
volume Y is 40 mM, then the reconstituted solution has a tonicity of 240 mOsm/L plus 80
mOsm/L, which is about isotonic, and which has a sufficient ionic strength to prevent
erythrocyte aggregation
[0022] In one aspect, the invention provides a method for preparing a lyophilized
Factor IX formulation for mtravenous mjection, the method comprising adding an about 25
mM to about 150 mM sodium chloride solution to the lyophilized Factor IX formulation
thereby resulting in a formulation prepared for mtravenous mjection, wherein the prepared
formulation is about isotonic with respect to plasma or is slightly hypotonic or slightly
hypertonic with respect to plasma, and wherein the prepared formulation has a sufficient
ionic strength to prevent erythrocyte agglutination In one aspect, the lyophilized Factor IX
formulation, when measured as if reconstituted in water, comprises (a) from about 5 mM to
about 30 mM histidine, (b) from about 0 1M to about 0 3M glycine, (c) from about 0 5 to
about 2 percent sucrose, (d) from about 0 001 to about 0 05 percent polysorbate, and (e) from
about 0 4 mg/mL to about 20 mg/mL of Factor IX, or from about 0 1 mg/mL to about 100
mg/mL or some other soluble amount of Factor IX, or from about 10 IU/mL to about 500
IU/mL of Factor K, or from about 10 IU/mL to about 5000 IU/mL of Factor IX In one
aspect, an about ^0 mM sodium chloride solution is added to the lyophilized Factor DC

formulation In one aspect, about 5 tnL of the about 40 mM sodium chloride solution is
added to the lyophilized Factor IX formulation In one aspect, the lyophilized Factor EX
formulation if measured as if it is reconstituted in water comprises about 10 mM histidine,
about 0 26M glycine, about 1% sucrose, and about 0 005% polysorbate
[0023] In one aspect, the invention provides a pharmaceutical kit comprising (a) a
vial containing a lyophilized cake, wherein if the lyophilized cake is reconstituted in about 5
mL of water the solution would comprise (I) from about 5 mM to about 30 mM histidine, (a)
from about 0 1M to about 0 5M glycine, (in) from about 0 5 to about 2 percent sucrose, (IV)
from about 0 001 to about 0 05 percent polysorbate, and (v) from about 0 4 mg/mL to about
20 mg/mL of Factor EX, or from about 0 1 mg/mL to about 100 mg/mL or some other soluble
amount of Factor EX, or from about 50 IU/mL to about 500 IU/mL of Factor EX, or from
about 10 IU/mL to about 5000 IU/mL of Factor EX, (b) a 25 mM to about 150 mM sodium
chloride solution, and (c) instructions for reconstituting the lyophilized cake with the sodium
chloride solution, such that after reconstitution the resultant solution is about isotonic and has
a sufficient ionic strength to prevent erythrocyte aggregation upon intravenous injection
[0024] In one aspect, the mvention provides a pharmaceutical kit comprising- (a) a
vial containing a lyophilized cake, wherein if the lyophilized cake is reconstituted in 4 mL of
water the solution would comprise (I) about 10 mM histidine, (n) about 0 26M glycme, (m)
about 1 percent sucrose, (iv) about 0 005 percent polysorbate 80, and (v) from about 50
IU/mL to about 5000 IU/mL of Factor IX, (b) an about 40 mM sodium chloride solution, and
(c) instructions for reconstituting the lyophilized cake in the vial with about 5 mL of an about
40 mM sodium chloride solution, such that after reconstitution the resultant solution
comprises (I) from about 7 or 8 to about 10 mM histidine, (u) from about 200 to about 210
mM glycme, (m) from about 0 7% to about 0 9% sucrose, (iv) about 0 004% polysorbate 80,
(v) from about 50 IU/mL to about 5000 IU/mL of Factor IX, and (vi) about 40 mM NaCl
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 shows erythrocyte sedimentation results from experiments described
in Example 3 Erythrocyte sedimentation was measured at 60 minutes using an adaptation of
the modified Westergren method (see Example 2), in which human blood collected in EDTA
was mixed 1 4 with test solutions After 60 minutes, the distance in mm between the zero
mark and tie erythrocyte plasma interface was measured Horizontal bars represent the mean

and vertical brackets the standard deviation fiom a total of 12 donors Results were
combined from 4 independent experiments each of which evaluated blood from 3 donors
[0026] Figure 2 shows an erythrocyte sedimentation evaluation on BeneFIX®
formulations reconstituted with NaCl solutions A 40 mM NaCl solution is sufficient to
prevent erythrocyte agglutination when used to reconstitute either the currently marketed
BeneFIX® product or a new formulation of BeneFIX® (BeneFIX®-R where prior to
lyophihzation the fill solution was comprised of 4 mL and had concentrations of 10 mM
histidine, 260 mM glycine, 1% sucr- ' 005% polysorbate 80, and after lyophihzation it
was reconstituted in 5 mL of 40 mM1 ^-'chloride such that after reconstitution
BeneFDC®-R comprises 40 mM NaCl, 8 mM histidine, 208 mM glycine, 0 8% sucrose, and
0 004% polysorbate)
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention provides methods for preparing pharmaceutical formulations for
injection (in particular, preparations readied for intravenous injection) that do not cause
erythrocyte agglutination, hemolysis, and/or cell shrinkage To prevent agglutination, a
pharmaceutical formulation ready for injection needs to have sufficient ionic strength To
prevent hemolysis or cell shrinkage, a pharmaceutical formulation ready for injection needs
to be about isotonic with respect to plasma The invention provides methods that prepare
pharmaceutical formulations for injection that have both the sufficient ionic strength to
prevent agglutination and the requisite tonicity to prevent significant hemolysis or cell
dehydration or shrinkage The present methods involve the use of sodium chloride solutions
that are about 25 mM to about 150 mM for reconstituting lyophihzed cakes (or other non-
hquid pharmaceutical formulations) into solution or for diluting pharmaceutical formulation
solutions Whether the sodium chloride solutions are used for reconstitution or for dilution,
the addition of particular sodium chloride solutions result in a pharmaceutical preparation for
injection that is about isotonic with respect to plasma or blood and is of sufficient ionic
strength to prevent erythrocyte aggregation upon injection, in particular, upon intravenous
injection
10028] Terms
[0029] As used herein, the "molality" of a solution is the number of moles of a solute
per kilogram of solvent

[0030] As used herein, the "molarity" of a solution is the number of moles of solute
per liter of solution
[0031] As used herein, an "osmole" is the amount of a substance that yields, in ideal
solution, that number of particles (Avogadro's number) that would depress the freezing point
of the solvent by 1 86K
[0032] As used herein, the "osmolality" of a solution is the number of osmoles of
solute per kilogram of solvent Osmolality is a measure of the number of particles present in
solution and is independent of the size or weight of the particles It can be measured only by
use of a property of the solution that is dependent only on the particle concentration These
properties are vapour pressure depression, freezing point depression, boiling point elevation,
and osmotic pressure, and are collectively referred to as colligative pioperties
[0033] As used herein, the "osmolality" of a solution is the number of osmoles of
solute per liter of solution
[0034] As used herein, a "pharmaceutical formulation" that is readied or prepared for
injection can be any drug intended to be administered into a subject For example, a
pharmaceutical formulation can be a lyophilized cake, a solution, a powder, or a solid The
formulation, if not in liquid form, is reconstituted into solution with aNaCl solution of the
invention If the formulation is in liquid form, the formulation is diluted or mixed with a
NaCl solution of the invention
[0035] Ionic Strength
[0036] Ionic strength is a characteristic of an electrolyte solution (a liquid with
positive and negatively charged ions dissolved in it) It is typically expressed as the average
electrostatic interactions among an electrolyte's ions An electrolyte's ionic strength is half of
the total obtamed by multiplying the molality (the amount of substance per unit mass of
solvent) of each ion by its valence squared
[0037] Ionic strength is closely related to the concentration of electrolytes and
indicates how effectively the charge on a particular ion is shielded or stabilized by other ions
(the so-called lonic atmosphere) in an electrolyte The main difference between ionic strength
and electrolyte concentration is that the former is higher if some of the ions are more highly
charged For instance, a solution of fully dissociated (broken down) magnesium sulfate
(Mg2+S042") has 4 times higher lonic strength than a solution of sodium chloride (Na+CT) of
the same concentration Another difference between the two is that ionic strength reflects the

concentration of free ions, and not just of how much salt was added to a solution Sometimes
a salt may be dissolved but the respective ions are still bound together pairwise, resembling
uncharged molecules in solution In this case the ionic strength is much lower than the salt
concentration
[0038] For the invention, pharmaceutical preparations are prepared for injection such
that they are not only isotonic, but also have a sufficient ionic strength to prevent RBC
agglutination A sufficient ionic strength of a preparation ready for injection (z e, a
lyophihzed cake that is reconstituted with a NaCl solution of the invention, or a drug solution
that is diluted with a NaCl solution of the invention) can have, foi example, at least about 25
milhequivalents per liter (mEq/L) of Na and CI" ions In one embodiment, a sufficient ionic
strength is at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at least about 40 mEq/L of
Na+ and CI" ions
[0039] Ionic strength can also be described in terms of the conductivity of a solution
Conductivity is the ability of a material or solution to conduct electric current The principle
by which instruments measure conductivity is simple - two plates are placed in the sample, a
potential is applied across the plates (normally a sine wave voltage), and the current is
measured Conductivity (G), the mverse of resistivity (R) is determined from the voltage and
current values accordmg to Ohm's law G = I/R = I (amps) / E (volts)
[0040] Since the charge on ions in solution facilities the conductance of electrical
current, the conductivity of a solution is proportional to its ion concentration In some
situations, however, conductivity may not correlate directly to concentration However, for
sodium chloride solutions, conductivity is directly proportional to ion concentration The
basic unit of conductivity is the Siemens (S), formerly called the mho Since geometry of a
test sample affects conductivity values, standardized measuiements are expressed in specific
conductivity units (S/cm) to compensate for variations in electrode dimensions Specific
conductivity (C) is simply the product of measured conductivity (G) and the electrode cell
constant (L/A), where L is the length of the column of liquid between the electrode and A is
the area of die electrodes C = Gx (L/A) If the cell constant is 1 cm", the specific
conductivity is the same as the measured conductivity of the solution Although electrode
shape vanes, an electrode can always be represented by an equivalent theoretical cell
[0041] Thus, in one embodiment, a sufficient ionic strength of a preparation ready for
injection can have, for example, at least about a conductivity of about 4 mS/cm or higher In

another embodiment, the sufficient ionic sttength of a solution ready for injection is at least
about2 5, 2 6, 2 7, 2 8, 2 9, 3 0, 3 1, 3 2, 3 3, 3 4, 3 5, 3 6, 3 7, 3 8, or atleast about 3 9
mS/cm
[0042] An operational definition of whether a solution has a sufficient ionic strength
to prevent erythrocyte agglutination can also be used to explain the term "sufficient ionic
strength " For example, this can be based on an experiment of adding a test solution to whole
blood and observmg the distance of erythrocyte sedimentation (see Example 2, adapted,
modified Westergren method) In one embodiment, if a test solution when mixed with whole
blood in a ratio of 4 1 provides an erythrocyte sedimentation at 60 minutes that is less than
about 10 mm (see Example 2, Figures 1 and 2, for example), then the test solution has a
sufficient ionic strength to prevent erythrocyte agglutination In another embodiment, if a
test solution when mixed with whole blood in a ratio of 4 1 provides an erythrocyte
sedimentation at 60 minutes that is less than about 5 mm, then the test solution has a
sufficient ionic strength to prevent erythrocyte agglutination
[0043] Solution Osmolanty
[0044] The methods of the invention provide pharmaceutical preparations for
injection that are about isotonic with respect to blood To determine whether a
pharmaceutical preparation is about isotonic with respect to blood, one calculates the
osmolanty for all chemical components of a solution including the diluent The osmolar
concentration of pharmaceutical preparations for injection (parenteral solutions) can exert
adverse effects on the blood cells and vessels of the human body Tonicity can be calculated
for fluids and dissolved or diluted medications, which are expressed in a numerical value of
milhosmoles per liter of fluid (mOsm/L) This value is also known as osmolanty The
osmolanty of blood ranges between 285 and 310 mOsm/L When hypotonic or hypertonic
solutions are injected into blood, fluid shifts into or out of cells, which can cause a vanety of
negative effects
[0045] Solution osmolanty is based in part on the concepts of osmosis and osmotic
pressure Osmosis is the diffusion of solutes (dissolved particles) or the transfer of fluid
through semipermeable membranes such as blood vessels or cell membranes Osmotic
pressure, which facilitates the transport of molecules across membranes, is expressed in
osmolar concentrations and is referred to as hypo-osmotic (hypotonic), iso-osmotic (isotonic),

or hypei-osmotic (hypertonic) when compared with biologic fluids such as blood or plasma
The term "tonicity" and "osmotic pressuie" are often considered to be synonymous
[0046] The osmotic pressure is the hydrostatic (or hydraulic) pressure required to
oppose the movement of water through a semipermeable membrane in response to an
'osmotic gradient' (i e, differing particle concentrations on the two sides of the membiane)
Serum osmolality can be measured by use of an osmometer or it can be calculated as the sum
of the concentrations of the solutes present in the solution The value measured in the
laboratory is usually referred to as the osmolality The value calculated from the solute
concentrations is reported by the laboratory as the osmolality The osmolar gap is the
difference between these two values
[0047] Herein, tonicity and osmotic pressure are to be considered synonymously, and
are to be understood broadly Tonicity can mean the effective osmolality and is equal to the
sum of the concentrations of the solutes in a solution that have the capacity to exert an
osmotic foice across a membrane, nicludmg a cell membrane In the strict sense, osmolality
is a property of a particular solution and is independent of any membrane Tonicity is a
property of a solution in reference to a particular membrane However, the invention shall
refer to solutions being isotonic with respect to biological solutions such as blood or plasma,
and this referencing shall include the meaning that the particular solution is isotonic with
blood or plasma with lespect to a cell membrane of a cell in the blood or plasma or other
biological solution [0048] An operational definition of tonicity can be used to explain the term This can
be based on an experiment of addmg a test solution to whole blood and observing the result
If the RBCs in whole blood swell and rupture, the test solution is said to be hypotonic
compared to normal plasma. If the RBCs shrink and become crenated, the test solution is said
to be hypertonic compared to normal plasma If the RBCs stay the same, the test solution is
said to be isotonic with plasma The RBC cell membrane is the reference membrane For
example, whole blood placed in normal saline (z e, 0 9% sodium chloride) will not swell, and
hence normal saline is said to be isotonic
[0049] Characteristics of Isotonic Solutions
[0050] The invention provides methods for preparing or readying pharmaceutical
formulations for injection into a subject, wherein the formulations are prepared into solutions
that are (1) about isotonic with respect to blood (oi plasma or other biologic fluid) or ate not

so hypertonic or hypotonic as to cause significant hemolysis, thrombosis, or vessel irritation,
and (2) have sufficient lonic strength to prevent erythrocyte aggregation
[0051] In one embodiment, isotonic (with respect to blood) pharmaceutical
formulations ready for injection have a tonicity or osmolality that is greater than about 270
mOsm/L and less than about 330 mOsm/L In one embodiment, the isotonic pharmaceutical
formulations ready for injection have a tonicity or osmolality that is greater than about 270
mOsm/L and less than about 328 mOsm/L
[0052] Although solutions such as 0 9% sodium chloride and 5% dextrose are
isotonic, when they are used to reconstitute or dilute many pharmaceutical formulations, the
resultant solution may not have sufficient ionic strength to pi event erythrocyte agglutination
(as for 5% dextrose) or may have too much lonic strength such that the resultant solution is
hypertonic Thus, the methods of the invention use solutions for dilution or reconstitution
that contain a minimum concentration of sodium chloride to provide (a) a sufficient ionic
strength to mitigate erythrocyte aggregation, and (b) a sufficient tonicity to prevent
hemolysis, and a maximum concentration of sodium chloride to provide (c) a resultant
tonicity that is not so great as to be hypertonic solutions Further, the methods of the
invention use solutions for dilution or reconstitution that can provide a sufficient ionic
strength and isotonicity with respect to blood, while also maintaining a practical injection
volume for the pharmaceutical preparation
[0053] Characteristics of Hypotonic Solutions
[0054] The invention provides methods for preparing pharmaceutical formulations for
injection into a subject, wherein the formulations are prepared into solutions that are not
hypotonic with respect to blood or aie not so hypotonic (i e, slightly hypotonic with respect
to blood) so as to cause significant hemolysis In one embodiment, the pharmaceutical
formulations ready for injection that is considered slightly hypotonic with respect to blood
can have a tonicity or osmolality that is less than about 270 mOsm/L and greater than about
240 mOsm/L In one embodiment, the pharmaceutical formulations ready for injection that is
considered slightly hypotonic with respect to blood can have a tonicity or osmolality that is
less than about 270 mOsm/L and greater than about 220 mOsm/L
[0055] Examples of hypotonic solutions include many pharmaceutical preparations
that are readied for injection with stenle water When hypotonic solutions are injected, a
fluid shift occurs and water is moved into the endothelial cells of the vem and blood cells

Cells that absorb too much water can buist, and thus, injection of hypotonic solutions can
cause vein irritation, phlebitis, and hemolysis
[0056] Characteristics of Hypertonic Solutions
[0057] The invention provides methods for preparing pharmaceutical preparations for
injection into a subject, wherein the preparations are prepared into solutions that aie not
hypertonic with respect to blood or are not so hypertonic (i e, slightly hypertonic) as to cause
significant thrombosis and/or vessel irritation In one embodiment, the pharmaceutical
formulations ready for injection that is considered to be slightly hypertonic can have a
tonicity or osmolality that is greater than about 340 mOsm/L and less than about 600
mOsm/L In one embodiment, the pharmaceutical formulations ready for injection that is
considered to be slightly hypertonic can have a torn city or osmolality that is gi eater than
about 340 and less than about 375, 400, 425, 450, 475, 500, or about 575 mOsm/L In
general, hypertonic solutions exhibit a tonicity that is greater than about 340 mOsm/L
Solutions with an osmolality that is greater than about 600 mOsm/L should be used with care in injections
[0058] Examples of undesirable hypertonic solutions include many pharmaceutical
formulations that are readied for injection with 10% dextiose, or pharmaceutical preparations
that have multiple additives that affect osmolality When hypertonic solutions are mjected, a
fluid shift occurs and water is drawn out of the endothelial cells of the vein and blood cells
Cells that lose too much water can shrink, and thus, injection of hypertonic solutions can
cause vein irritation, phlebitis, and thrombosis
[0059] pH
[0060] The pH of blood ranges from about 7 35 to about 7 45, which is considered
neutral Pharmaceutical preparations with a pH value below 7 are considered acidic drugs
and those with a pH value below 4 1 are considered very acidic Drugs with a pH value
higher than 7 5 are considered basic or alkaline drugs, and those with a pH value higher than
9 0 are considered very basic or alkaline Very acidic or very alkaline drug solutions can
cause phlebitis and thrombosis Thus, in one embodiment, the invention provides sodium
chloride solutions for reconstituting lyophihzed drug formulations or for diluting liquid drag
formulations to ready these formulations for intravenous injection, wherein the readied
preparations for injection (1) do not have a pH that is less than 4 1 or greater than 9 0, (2)
have a sufficient ionic strength to prevent erythrocyte aggregation, and (3) is about isotonic

(or slightly hypertonic or hypotonic) with respect to blood such that hemolysis or crenation
does not occur to RBCs However, the pH of a solution is not a consideration with respect to
whether the solution has a sufficient ionic strength to prevent erythrocyte aggiegation In
other words, if a lyophihzed formulation when reconstituted with water has a pH that is
below 4 1 or greater than 9 0, this does not prevent the use of a sodium chloride solution for
reconstitution in order to prevent erythrocyte agglutination
[0061] Calculating Osmolality
[0062] The osmolality of any pharmaceutical preparation can be calculated by using
the following formula Osmolality = (weight of substance (g) divided by the molecular
weight of the substance (g/L)) multiplied by the number of species multiplied by 1000 for
milliosmolanty The term "species" refers to the number of ions or chemical species formed
when dissolution occurs
[0063] Pharmaceutical Formulations Ready or Prepared for Iniection
[0064] The invention provides methods for reconstituting lyophihzed drug products
into solution in order to prepare the drug product for injection mto a subject The
reconstituted drug product is ready for injection by having a sufficient ionic strength and a
tonicity that is about isotonic with respect to blood
[0065] The methods of the invention also pertain to diluting drug solutions in order to
prepare the drug solution for injection mto a subject The diluted drug solution is ready for
injection by having a sufficient lonic strength and by being about isotonic with respect to
blood
[0066] In one embodiment, the lyophihzed or dry drug product/formulation, if
reconstituted in water, has an osmolality of about 100 mOsm/L to about 360 mOsm/L
Because the mvention provides methods for reconstitution using about 25mM to about 150
mM sodium chloride solutions, the practitioner should use a particular sodium chloride
solution based upon the expected combined osmolality of the lyophiiized drug product
reconstituted into the sodium chloride solution Thus, for example, if a lyophiiized drug
product if reconstituted in water has an osmolality of about 300 mOsm/L, then the NaCl
solution for reconstitution should be about 25 mM to about 30 mM In another example, if a
lyophiiized drug product if reconstituted in water has an osmolality of about 100 mOsm/L,
then the NaCl solution for reconstitution should be about 25 mM to about 130 mM

[0067] In one embodiment, a drug product (whether lyophilized or in solution) to be
prepared for injection by the present methods does not contam HBS (hydroxyethyl starch)
HES-containing formulations, despite reconstitution or dilution with NaCl solutions for ionic
strength, can cause erythrocyte sedimentation and agglutination in in vitro experiments In
another embodiment, a drug product to be prepared for injection by the present methods does
not contain dextrans, because adapted modified Westergren methods (see Example 2) show
that the addition of NaCl would not counteract the effect of enhanced sedimentation that
dextrans can cause
[0068] In one embodiment, the invention provides methods for preparing a
pharmaceutical preparation for intravenous injection wherein the pharmaceutical preparation
is a lyophilized cake comprising a primary bulking agent The primary bulking agent can be,
for example, mostly non-ionizing Non-ionizing bulking agents include, but are not limited
to, mannitol, glycme, sucrose, lactose, other disacchandes, therapeutic proteins or the active
ingredient of a formulation itself, or other bulking agents known to one skilled in the art The
concentrations of non-ionizing bulking agents do not significantly affect whether a solution
has a sufficient lonic strength to prevent agglutination However, therr concentrations do
have an effect on osmolality, and therefore, then concentrations can have an effect on
torn city The concentration of glycme, for example, is equivalent to its contribution to
osmolality, thus 10 mM glycine is equivalent to 10 mOsrn/L
[0069] Protein mgredients in a drug formulation, including active ingredients, do not
significantly affect ionic strength of a solution or the osmolality of a solution For example,
assume a molecular weight of 50,000 for a protein and assume 2 5 mg of the protem in a 1-2
mL solution to be injected This is equivalent to 0 05 mM, thus, the protein does not
appreciably contribute to osmolality
[0070] Pharmaceutical preparations also often contain surfactants, such as
polysorbate-80 Polysorbate-80 and other surfactants are large molecular weight molecules,
so amounts that are usually present in preparations, such as 0 001% to 0 01%, are too small to
contribute appreciably to osmolality or ionic strength Other surfactants include Bnj® 35,
Bnj® 30, Lubrol-px™, Triton X-10, Pluronic® F127, and sodium dodecyl sulfate (SDS)
[0071] Other large molecular weight molecules that may be present in small amounts
m pharmaceutical formulations that do not appreciably affect osmolanty or lonicity are
polymers, with the,qualification that they are not salts like dextran sulfate. Exemplary

polymers include dextran, poly(vmyl alcohol) (PVA), hydroxypropyl methylcellulose
(HPnic), gelatin, polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP)
[0072] Pharmaceutical formulations also often contain sucrose or other sugars or
polyols, oftenin an amount of 0-2, 0-5, or 0-10% or higher For example, 5-10% sucrose can
be used when the formulation does not comprise a bulking agent Generally, where a
formulation is lyophihzed, and amounts of the formulation are identified herein as
percentages or molarity amounts, this is in refeience to percentages and molarity of a solution
prior to lyophihzation (z e, fill amounts) Thus, when a solution has 1% sucrose, this is
equivalent to 29 2 mM Because sucrose does not appreciably ionize or disassociate in solution, 29 mM of sucrose is equivalent to 29 mOsm/L Other sugars or polyols that do not
appreciably ionize or disassociate in solution include glycerol, xyhtol, sorbitol, mannitol (also
can be used as a bulking agent), glucose, inositol, raffinose, maltotnose, lactose, and
trehalose
[0073] Pharmaceutical formulations also can contain buffering agents Buffering
agents include, for example, acetate, citrate, glycine, histidine, phosphate (sodium or
potassium), diethanolamme and Tns Buffering agents include those agents that maintain a
solution pH in an acceptable range Buffering agents such as glycine, histidine, and
diethanolamme are mostly non-ionizing, and thus therr concentrations are equivalent to
osmolality and should be kept in mind when considering whether a formulation ready for
injection is about isotonic Buffering agents such as acetate and citrate are usually salts, and
are therefore ionizing, and therr concentrations are multiplied with respect to calculating therr
contribution to a solution's osmolanty
[0074] Pharmaceutical formulations can include essentially any active ingredient,
including protems, nucleic acids, viruses, and chemical compounds These molecules mostly
do not appreciably affect the ionic strength or the osmolanty of a solution to be injected If
these molecules are salts, as often small molecule compounds are pharmaceutical salts, then
they may appreciably affect the lonic strength and/or osmolanty
[0075] Certain ammo acids are also found in some pharmaceutical formulations and
are used as cryoprotectants, lyoprotectants and/or bulking agents Some ammo acids, such as
histidine, are mostly non-ionizing, and thus the concentration of an amino acid in a
formulation should only be kept in mind when calculating the osmolanty of a solution such
that a formulation ready for injection is about isotonic with respect to blood

[0076] BeneFIX®
[0077] BeneFIX® is produced by a genetically engutieeied Chinese hamster ovary
(CHO) cell line that is extensively characterized and shown to be free of infectious agents
The stored cell banks are free of blood or plasma products The CHO cell line secretes
recombinant Factor DC (rFIX) into a defined cell culture medium that does not contain any
pioteins denved from animal or human sources, and the recombinant Factor DC is purified by
a chromatography purification process that does not require a monoclonal antibody step and
yields a high-punty, active product A membrane filtration step that has the ability to letain
molecules with apparent molecular weights >70,000 (such as large proteins and viral
particles) is included for additional viral safety BeneFIX® is predominantly a single
componentbySDS-polyacrylamidegel electrophoresis evaluation The potency (in
international units, IU (IU)) is determined using an in vitro one-stage clotting assay against
the World Health Organization (WHO) International Standard for Factor DC concentrate One
international unit is the amount of Factor DC activity present in 1 mL of pooled, normal
human plasma The specific activity of BeneFDC® is greater than oi equal to 180 IU per
milligram of protein BeneFDC® is not derived from human blood and contains no
preservatives or added animal or human components
[0078] BeneFIX® is formulated as a sterile, nonpyrogenic, lyophihzed powder
preparation BeneFDC® is mtended for intiavenous (IV) injection It is available in single use
vials containing the labeled amount of Factor DC activity, expressed in international units
(IU) Each vial contains, for example, nominally 250, 500, or 1000 IU (or more, including
2000 IU) of coagulation Factor DC (Recombinant) After reconstitution of the lyophilized
drug product with sterile water, the concentrations of exciprents in the 500 and 1000 IU
dosage strengths are 10 mM L-histidine, 1% sucrose, 260 mM glycine, 0 005% polysorbate
80 The concentrations after reconstitution in the 250 IU dosage strength are half those of the
other two dosage strengths The 500 and 1000 IU dosage strengths are isotonic after
reconstitution, and the 250 IU dosage strength has half the tonicity of the other two dosage
strengths after reconstitution All dosage strengths yield a cleai, colorless solution upon
reconstitution
[0079] In one embodiment, the invention provides methods for preparing BeneFDC®
to be injected into a subject, where BeneFDC® is reconstituted into a sodium chloride
solution, wherein the sodium chloride solution is greater than about 25 mM and less than
about 150 mM In one embodiment, the sodium chloride solution used to reconstitute

BeneFIX® is about 40 mM In one embodiment, one vial of lyophilized BeneFIX® is
reconstituted into about 4-5 mL of a 25mM-150mM sodium chloride solution
[0080] Reformulated BeneFIX® (BeneFLX®-R)
[0081] Because of the low ionic strength of BeneFIX® reconstituted in water, various
reformulations were made (Reformulated BeneFIX® (BeneFIX®-R)) The goal was to add
sufficient ionic strength to the BeneFIX® formulation so as to mitigate the potential for RBC
agglutination In order to increase the ionic strength of BeneFIX®, sodium chloride can be
incorporated into the reconstituted product by replacing the sWFI as the reconstituting
solution Alternatively, BeneFIX® can be reformulated by adding NaCl to the pre-
lyophilization formulation, such that reconstitution can still be achieved with water, but
lyophilizing salt solutions is more difficult, and it is more practical to simply reconstitute
lyophilized cakes with NaCl solutions This is also true for other lyophihzed pharmaceutical
preparations that do not have a sufficient lonic strength when reconstituted with sWFI to
prevent agglutination
[0082] In one embodiment, BeneFIX®-R can be lyophihzed in the same formulation
as BeneFIX® (10 mM L-histidine, 260 mM glycine, 1% sucrose, 0 005% polysorbate 80),
and only the rFIX concentration will differ BeneFlX®-R bulk drug product can then be
filled, for example, at 4 mL per vial with 10 rnM L-histidine, 260 mM glycme, 1% sucrose,
0 005% polysorbate 80, and lyophihzed When BeneFIX®-R is reconstituted to 5 mL per
vial with a 25-150 mM NaCl solution, such as with 40 mM NaCl, the concentration of the
exciprents is reduced by 20% when compared to the current BeneFIX® formulation pre-
lyophihzation This strategy lesults in a formulation that is (1) isotonic, (2) has sufficient
ionic strength to reduce the potential for RBC agglutination, and (3) reduces the injection
volume for higher doses of rFIX BeneFIX®-R can be provided with, for example, 250, 500,
1000, and 2000IU of rFIX per vial dosage strengths Thus, after leconstitution with 5 mL of
a 25-150 mM NaCl solution, the resultant BeneFLX®-R formulation solution can be from
about 7 to about 9 mM histidine, from about 188 to about 220 mM glycine, from about 0 7%
to about 0 9% sucrose, and about 0 004% polysorbate 80 Depending upon the amount of
rFIX in a vial, for example, 250, 500,1000, or 2000 IU, reconstitution in 5 mL of a NaCl
solution would result in an rFIX concentration of about 50,100,200, or 400 IU/mL,
respectively The osmolality of the BeneFIX®-R formulation reconstituted in 5 mL of a 40
mM NaCl solution is about 320 mOsm/L

[0083] Exemplary Formulations To Be prepaied for Imection
[0084] Recombinant Factor IX can also be lyophihzed in formulations described in
U S Patent No 6,372,716, which is hereby incorporated by reference for all purposes,
including the formulations described therein The formulations described in the patent can be
used with the present invention, where the formulations are not limited to having rFIX as the
active ingredient Thus, for example, lyophihzed formulations that can be reconstituted with
25 mM-150 mM sodium chloride solutions to prepare them for injection, include
formulations that comprise glycine, polysorbate, sucrose, histidine, and an active ingiedient
The active ingredient can be essentially any protein, virus, nucleic acid, or chemical
compound, for example The glycine can have a concentration, for example, of about 0 1M
to 0 3M The polysorbate can have a concentration, for example, of about 0 001 to about
0 05% The sucrose can have a concentration, for example, of about 0 5% to about 2% The
histidine can have a concentration, for example, of about 5 mM to about 30 mM
[0085] In one embodiment, a lyophihzed formulation to be reconstituted with 25-150
mM sodium chloride solution comprises about 0 1 to 0 3M glycine, about 0 5 to 2% sucrose,
0 001 to about 0 05% polysorbate, about 5 to about 30 mM histidine, and about 0 1 to about
20 mg/mL of an active ingredient The active ingredient can be, for example, IFIX In
another embodiment, a iFIX lyophihzed formulation to be reconstituted with 25-150 mM
sodium chloride solution comprises about 0 13 to about 3 mg/ml rFDC or about 50 to about
600 IU/mL of rFIX, about 0 26M glycine, about 10 mM histidine, about 1% sucrose, and
about 0 005% polysorbate
EXAMPLES OF THE INVENTION
[0086] The examples described below are provided to illustrate aspects of the present
invention and are not included for the purpose of limiting the invention
Example 1 Effects of Anti-Coagulants and Formulation Components on BeneFDC®-
Associated RBC Agglutination
[0087] There are occasional reports of BeneFIX®-associated RBC agglutination in
butterfly catheter lines and syringes Recent studies in blood from hemophilia dogs
demonstrate that the agglutination occurs in the absence of recombmant human FIX in the
formulation buffer Thus, the present study investigated whether standard anti-coagulants,
various BeneFDC® components, and ionic strength affects the BeneFIX®-associated
agglutination phenomenon

[0088] Blood was obtained from a pool of anonymous human volunteers and was
collected into Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing a standard
anti-coagulant, such as ethylenediamine tetra-acetic acid (EDTA), sodium citrate, or heparin
[0089] To test for agglutination, the blood samples in the Vacutamer tubes were first
continuously mixed on a nutator Blood, 12 5 |J.L, was diluted in 87 5 |iL of a test solution in
a 48-well cell culture plate, and was allowed to incubate at room temperature for 2 minutes
prior to observation under an inverted-phase contrast microscope at 400x magnification
Each well was videotaped to capture stall images in order to score RBC agglutination
according to the following criteria listed below in Table 1

[0090] Effects of Standard Anti-Coagulants on RBC Agglutination The first study
included the assay of blood samples from three donors Blood was collected into EDTA,
heparin, and sodium citrate Vacutamer tubes for each donor The test articles included
BeneFTX®, 100 IU/mL, reconstituted in each of the following NaCl concentrations 0 mM
(sWFI), 20 mM, 40 mM, 60 mM, and 77 mM NaCl In addition, dextrose 5% in water
(D5W) was included as a positive control The samples were diluted at a blood to BeneFK®
or blood to D5W ratio of 1 8 Images were captured, stored digitally, masked, and scored for
agglutination Two minutes following the addition of blood to the BeneFIX® reconstituted
with sWFI, agglutination was seen in the samples from all three donors regardless of the type
of anti-coagulant used As increasing concentrations of NaCl were used, the agglutination
response was attenuated Marked agglutination was seen when blood was added to D5W
[0091] Effects of NaCl on RBC Agglutination in Pre-screenedDonojs- Donor
samples were collected into heparmized Vacutainer tubes Because spontaneous

agglutination had been observed in some previous samples, all donors were initially screened
usmg 154 mM NaCl If agglutination was observed, the sample was disqualified fiom further
assays The remaining samples from each donor were diluted 1 8 into one of two
formulations (1) 100 IU/mL BeneFJK® reconstituted with sWFI, and (2) 100 lU/mL
BeneFIX® reconstituted with 154 mM NaCl Images were captured, stored digitally, masked
and scored for agglutination Agglutination was seen with BeneFIX® reconstituted sWFI,
whereas reconshtution with 154 mM NaCl reduced or eliminated the agglutination reaction
(see Table 2 for scores)

[0092] Effect of Formulation Components and BeneFIX® Concentration Several
formulations of BeneFIX® were evaluated to ascertain the impact of different components on
RBC agglutination Eight different mixtures, with and without 154 mM NaCl were prepared,
for a total of 16 mixtures (Table 3) Twelve donors were screened for spontaneous
agglutination and two were disqualified from further study Blood samples from five of the
ten remaining donors were used for the first 8 formulations (samples 1-A through 1-H) and
the other five donor samples were used for the second 8 formulations (samples 2-1 through 2-
P) Samples were diluted 1 8 as before and images were captured and scored as previously
described " '



[0093] Agglutination scores for the experiment laid out in Table 3 are shown below in
Tables 4A and 4B No particular component, whether the active ingredient or an exciprent,
of the BeneFDC® preparation was associated with agglutination As expected, removal of
glycine caused RBC lysis due to hypotonicity of the solution However, reconstitution with
154 mM NaCl leduced or eliminated the in vitro agglutination and the lysis
Table 4A Agglutination Scores - Varying the BeneFDC® Concentration

[0094] Effects of Lower Concentrations of NaCl on RBC Agglutination in Pre-
Sct eened Donors Donor samples were collected mto hepannized Vacutamer tubes

Because agglutination had been observed in some previous samples, all donors weie initially
screened with 154 mM NaCl If agglutination was observed, the sample was disqualified
from further assays The remaining samples from each donor were diluted 1 8 as before into
one of three formulations (1)100 IU/mL B eneFLX® with sWFI, (2) 100 IU/mL BeneFK®
with 40 mM NaCl, or (3) 100 IU/mL BeneFIX® with 77 mM NaCl Images were captured,
collected and scored as before The results aie provided in Table 5 below Agglutination was
seen in all five samples with BeneFIX® reconstituted with water, whereas reconstitution of
BeneFIX® with 40 mM or 77 mM NaCl reduced or eliminated the agglutination reaction

[0095] Analysis RBC agglutination occurred in blood anti-coagulated with heparin,
EDTA, or sodium citrate when mixed with BeneFIX® at a blood to reconstituted BeneFIX®
volume ratio of 1 8 No effect of anti-coagulant type was seen on the agglutination, therefore
heparin was used as the anti-coagulant in subsequent evaluations In some samples,
agglutination occurred in the presence of 0 9% NaCl alone (negative control for
agglutination) Therefore, subsequent evaluations were conducted only with samples that
exhibited no agglutination with the 0 9% NaCl control
[0096] The effects of three concentrations of BeneFIX® (100, 300, or 600 IU/mL)
and of each individual component of BeneFIX®, reconstituted in sWFI or NaCl solution
were then evaluated for RBC agglutination when mixed with heparin anti-coagulated blood at
a blood to BeneFIX® volume ratio of 1 8 Elimination of any particular component,
including the recombinant human FIX protein had no effect on the agglutination response
This indicates that a broad range of different pharmaceutical formulations can be readied
(reconstituted or diluted) with NaCl solutions for intravenous injection Removal of glycine
resulted in RBC lysis However, the use of 40 mM (0 234% NaCl, injectable), 77 mM

(0 45% NaCl, injectable) or 154 mM NaCl (0 9% NaCl, injectable) for reconstitution reduced
or eliminated the agglutination response and lysis
[0097] Thus, these results show that no particular component of the BeneFIX®
formulation is related to the observed agglutination Although removal of glycine from the
BeneFIX® formulation and reconstitution with water results in a hypotonic solution that
causes lysis, osmolality and tonicity is a distinct concern from ionic strength and
agglutination The results show that agglutination is associated with the low ionic strength of
the reconstituted recombinant Factor DC (rFIX) in sWFI, and reconstitution with NaCl
attenuates or eliminates agglutination Thus, reconstitution of other pharmaceutical
formulations with NaCl solutions, even NaCl solutions having a concentration below 154
mM, can prevent both agglutination and lysis upon mtravenous injection
Example 2 Adaptation of the Modified Westergren Method of Erythrocyte Sedimentation
Rate Measurement to Assess Erythrocyte Aggregation Induced by Pharmaceutical Agents or
Formulations
[0098] The currently marketed BeneFIX® formulation is a non-ionic formulation
During administration of the BeneFIX® formulation reconstituted in sWFI, there has been
mfiequent observations of erythrocyte aggregation (z e, agglutination) when a patient's blood
is mixed with the reconstituted BeneFIX®, such as in intravenous tubing The invention
provides the discovery that erythrocyte aggregation is associated with the formulation, not
rFIX, and is prevented by using diluents that contain at least about 40 mM NaCl To assist in
the design of custom diluents containing at least about 40 mM NaCl, a quantifiable assay was
designed that can be used to measure erythrocyte sednnentation, which is an established
method to assess erythrocyte aggregation in vitro The modified Westergren method, in
which blood is diluted 4 5m normal saline, was adapted to assess aggregation in blood that
had been diluted 1 4 or 1 8 with either saline or test solutions (i e, custom diluents)
[0099] Human blood was obtained from healthy adult volunteers and collected into
tubes containing EDTA To demonstrate the suitability of blood samples for sedimentation
experiments, the clinically defined erythrocyte sednnentation rate ESR.60 was measuied by the
modified Westergren method Briefly, well-mixed blood was diluted 4 5 with 154 mM NaCl,
gently mixed well, and then loaded immediately onto a self-zeroing Westergren tube that had
been placed in a custom 10-tube rack The distance in mm from the zero mark at the top of
the tube to the plasma erythrocyte interface after 60 minutes was measured and recorded
The ESRgo results were compared with published normal reference values (Morns, in W et

al, Basic examination of blood, in Henry, J B , ed , Clinical Diagnosis and Management by
Laboratory Methods, Philadelphia, PA, WB Saunders, 2001 479-519)
[00100] Two experiments were conducted to show the suitability of the
adapted, modified Westergren method to assess erythrocyte aggregation associated with
pharmaceutical formulations Human blood samples were suitable foi use in these assays
because all had ESB.60 values that were within normal limits These samples were diluted 1 4 in the first experiment and diluted 1 8 in the second
[0100] In the first experiment, erythrocyte sedimentation was enhanced by 5%
dextian 70 or BeneFIX® reconstituted with sWFI or 10 mM NaCl Within 5 mmutes,
aggregates were visible in the tubes that had been loaded with blood diluted with either 3%
dextran 70 or BeneFIX® reconstituted in sWFI By 60 minutes, erythrocyte sedimentation in
these tubes was markedly enhanced when compared with saline control (Table 6) By 90
mmutes, blood from one donor that was diluted in BeneFIX® reconstituted in 10 mM NaCl
had a two-phase sedimentation pattern with a clear erythrocyte plasma interface located 8
mm from the zero-mark and another interface between densely packed and less densely
packed erythrocytes located 149 mm from the zero mark

[0101] In the second experiment, erythrocyte sedimentation was enhanced by 5%
dextrose, "MFR-927" (the formulation buffer for BeneFIX® that lacks rFIX), and BeneFIX®
reconstituted with 10 mM NaCl (Table 7) Unlike the first experiment, sedimentation m

these tubes was rapid with maximal or neatly maximal sedimentation leached by 15 to 30
minutes

[0102] The results of these experiments demonstrate that the modified Westergren
method used to measure erythrocyte sedimentation can be adapted to assess erythrocyte
aggregation induced by pharmaceutical preparations or formulations For example,
measurement of erythrocyte sedimentation in Westergren tubes 60 minutes after loading
blood diluted 1 4 with test solutions is sufficient to distinguish between agents that enhance
aggregation (i e, 5% dextrose, 3% dextran 70, MFR-927, and BeneFIX® reconstituted in
sWFI) from those that do not (z e, saline and BeneFIX® reconstituted in diluents containing
about 25 mM or more NaCl)
Example 3 Adequacy of 40 mM NaCl in Diluent for Reformulated BeneFIX® to '
Ameliorate Formulation-Associated Erythrocyte Aggregation
[0103] The non-ionic formulation of currently marketed BeneFIX® has been
associated with in vitro erythrocyte aggregation, which can occur during administration when
patient blood is mixed with BeneFIX® in intravenous tubing The invention provides the
discovery that erythrocyte aggregation is associated with the formulation, not recombinant

Factor IX, and aggregation can be prevented by reconstituting BeneFIX® with diluents that
contain at least 40 mM NaCl
[0104] A goal of this Example is to test the robustness of a 40 rnM diluent for
reconstituting reformulated BeneFIX® (BeneFDC®-R) preparations Specifically,
experiments were designed to determine whether NaCl concentrations that deviate from 40
mM by as much as 10% are sufficient to prevent formulation-associated erythrocyte
aggregation, which was assessed by the adapted, modified Westergren method to measure
erythrocyte sedimentation rate (ESR) In addition, the robustness of the NaCl concentration
on erythrocyte sedimentation was assessed in both high (z e, 2000 IU) and low (z e, 250IU)
dose vials of BeneFIX®-R
[0105] The effect of BeneFIX®-R formulations, MFR-927, and 3% dextran 70 on
erythrocyte sedimentation was measured at room temperatuie using an adaptation of the
modified Westergren method, as described in Example 2 The study design is outlined in

[0106] On the day of an experiment, both high (vials of lyophilized B eneFDC®-R
containing ~2000 IU rFIX) and low dosage vials of BeneFIX®-R (vials of lyophihzed
BeneFIX®-R containing ~250 IU rFIX) were reconstituted immediately before use with 5
mL of 36 mM, 40 mM, or 44 mM NaCl The positive control solution 3% (w/v) dextran 70
was prepared in sterile Dulbecco's modified, calcium- and magnesium-free phosphate-
buffered saline (PBS-CMF, pH 7 4) Human blood was obtained from donors and collected

into Vacutainer tubes containing EDTA Blood samples were used for erythrocyte
sedimentation expenments within 3 hours of collection
[0107] Whole blood was diluted 1 4 in test solutions (z e, 400 pL of whole blood was
added to 1 2 mL of test solution), mixed well, and then loaded into self-zeroing disposable
glass Westergren tubes that were held absolutely vertical in a dedicated custom rack
Erythrocyte sedimentation after 60 minutes, which is the distance in mm from the zero mark
at the top of the tube to the plasma-erythrocyte interface, was measured and recorded
[0108] Results from all 12 donors were similar Currently marketed BeneFIX®
formulation buffer that lacked rFIX (MFR-927) and 3% dextran 70, a standard positive
control solution, displayed enhanced erythrocyte sedimentation as compared to blood that had
been mixed in NaCl test solutions (see Figure 1) Regardless of NaCl concentration in the
buffer (36 mM, 40 mM, or 44 mM) or rFIX concentration in the product, BeneFIX®-R did
not enhance erythrocyte sedimentation as long as it was reconstituted with a NaCl diluent
[0109] The results from this study demonsti ate that NaCl concentrations 10% higher
or lower than &0 mM in the BeneFIX®-R diluents are sufficient to prevent enhanced
erythrocyte sedimentation or aggregation (agglutination) Results also show that the
ameliorating effect of NaCl on formulation-associated erythrocyte aggregation was not
affected by the concentration of the active ingredient (z e, rFIX)
i
Example 4 Role of Ionic Strength in the Buffer for Reconstituting BeneFIX® in Causing
RBC Agglutination
[0110] Human blood was collected by venipuncture from four different donors into
standard heparrnized collecting tubes Formation of RBC agglutinates was tested by drawing
up 2 6 mL of buffer or reconstituted BeneFIX® protein into syringes followed by 0 6 mL of
heparrnized blcod The mixing/sedimentation behavior of the agglutinates was observed in
these syringes over time and photographically recorded usmg a digital camera
[0111] The composition of the current BeneFIX® formulation pre-lyophilization is
rFIX, about 10 mM hisudine, about 260 mM glycine, about 1% sucrose, and about 0 005%
polysorbate-80, pH 6 8 The BeneFIX® formulation is lyophihzed, and pnor to injection,
reconstituted in sWFI Originally, the sucrose was suspected to be the cause of agglutination
Hence, test buffers were formulated with 1 %, 0 5%, or 0% sucrose Each sucrose test buffer
was drawn up in syringes, followed by a small amount of hepannized blood The
agglutination of RBCs and subsequent rapid sedimentation were found to be identical for the

three buffers, suggesting that the sucrose content of the BeneFIX® formulation did not
account for the observed agglutination BeneFIX® reconstituted in normal saline, rather than
in sterile water, caused no agglutination These data indicate that the lonic strength of the
BeneFIX® formulation is not sufficient to prevent agglutination when BeneFIX® is
reconstituted in water
[0112] In order to determine the minimum lonic strength needed to eliminate
agglutination, BeneFIX® was reconstituted (or the pre-lyophihzation formulation of
BeneFIX® can be varied to include sodium chloride, although methods for lyophilizing
sodium chloride formulations is more difficult as compared to lyophilizing formulations that
do not have sodium chloride) with solutions having varying concentrations of NaCl (0, 10,
15, 20, 25, 30, 35, 40, 50, 75,100, and 137 mM) BeneFIX® was also reconstituted in
normal saline containing 154 mM NaCl RBC agglutination and sedimentation behavior was
examined in all of these solutions Table 9 shows the osmolality and ionic strengths
(expressed as conductivity) of some of these solutions along with other common intravenous
solutions Upon addition of sodium chloride to the BeneFIX® formulation, whether by
straight addition pnor to lyophilization or by reconstitution of a lyophihsate with a solution of
sodium chloride, the NaCl would be expected to fully dissociate to an equivalent
concentration of Na and CI" ions Thus, the ionic strength (expressed in mEq/L
(milhequivalents/L) of Na+ and CI") of a formulation buffer plus NaCl would be predicted to
be equivalent to the NaCl concentration if the formulation buffer does not contam other ions



[0113] Upon contact of blood with the solutions in Table 9 in syringes, RBC
agglutination was individually observed and then the syringes weie gently mixed and
inverted to observe blood settling Agglutinate formation was not observed in all
formulations of BeneFIX® reconstituted with at least about 40 mM NaCl (note in the prior
Example, about 36 mM NaCl was also sufficient to prevent agglutination) The behavior of
blood cells in these solutions containing at least 40 mM NaCl was indistinguishable from that
ui normal saline
[0114] Blood sedimentation was tested in a series of syringes containing BeneFIX®
formulation diluted (if pre-lyophihzation)/reconstituted (if lyophilized) in decreasing
concentrations of NaCl (starting at 40 mM) 15 minutes after inversion There was a
consistent concentration-dependent response in terms of agglutinate formation and
subsequent speed of sedimentation - increasing agglutination and quicker sedimentation with
decreasmg concentration of NaCl in the buffer I Obvious agglutination was visible with one
blood sample in buffer containing <25 mM NaCl and with another blood sample in buffer
containing <30 mM NaCl In all buffeTpTreparations with >40 mM NaCl, the behavior of
blood cells was indistinguishable from that in normal saline or BeneFIX® reconstituted in
normal saline BeneFIX® reconstituted with 40 mM NaCl corresponds to a calculated
conductivity of 4 mS/cm
[0115] The 5% dextrose injection solution, each mL of which contains 50 mg hydrous
dextrose USP in water for injection - has a calculated osmolality of 250 mOsm/L and a
calculated ionic strength of 0 mS/cm This solution, which is commonly administered
intravenously, also caused RBC agglutination and very rapid blood sedimentation similar to
that observed with BeneFIX® formulation reconstituted in water
[0116] Thus, these results indicate that RBC agglutination caused by BeneFIX®
reconstituted in water is due to the BeneFIX® formulation's low ionic strength (0 mEq/L
calculated ionic strength, <0 2 mS/cm measured conductivity) This problem can be
corrected by reconstituting in NaCl solutions having >40 mM NaCl such that the
reconstituted solution for injection has a calculated ionic strength of about 40 mEq/L (the
sufficient ionic strength to prevent agglutination can also be calculated as a conductivity of
about 4 mS/cm or higher) or higher

We claim
1 A method for preparing a Factor IX formulation for intravenous injection, the method
comprising adding a 25 mM to 150 mM sodium chloride solution to a lyophilized Factor IX
formulation thereby resulting in a formulation prepared for intravenous injection, wherein the
lyophilized formulation, if reconstituted in water, does not contain more than 5 mM of an
ionizing salt, and, wherein the prepared formulation
is isotonic with respect to plasma and has an osmolanty that is from 270 mOsm/L to 330
mOsm/L or
is slightly hypotonic with respect to plasma and has an osmolanty that is from 220 mOsm/L
to 270 mOsm/L or,
is slightly hypertonic with respect to plasma, and has an osmolanty that is from 330 mOsm/L
to 600 mOsm/L and,
wherein the prepared formulation has a sufficient ionic strength to prevent erythrocyte
agglutination upon intravenous injection
2 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
that is at least 25 mEq/L of Na+ and CI ions
3 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
that is at least 30 mEq/L of Na+ and CI ions
4 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
that is at least 36 mEq/L of Na+ and CI" ions
5 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
that is at least 40 mEq/L of Na+ and CI ions
6 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
that is at least 40 mEq/L of Na+ and CI ions and less than 150 mEq/L of Na+ and CI ions
7 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
as measured in conductivity that is at least 2 5 mS/cm
8 The method as claimed in claim 1, wherein the prepared formulation has an ionic strength
as measured in conductivity that is at least 4 0 mS/cm

9 The method as claimed in any one of claims 1 to 8, wherein the sodium chloride solution is
40 mM to about 150 mM
10 The method as claimed in any one of claims 1 to 8, wherein the sodium chloride solution
is 36 mM to 44 mM
11 The method as claimed in any one of claims 1 to 8, wherein the sodium chlonde solution
is 40 mM
12 The method as claimed in any one of claims 1 to 11, wherein the lyophilized formulation,
if reconstituted in water, does not contain more than 25 mM of an ionizing salt
13 The method as claimed in any one of claims 1 to 12, wherein prior to the addition of the
sodium chloride solution, the pharmaceutical formulation comprises histidine, glycine,
sucrose, and polysorbate
14 The method as claimed in any one of claims 1 to 13, wherein prior to the addition of the
sodium chloride solution, the pharmaceutical formulation if reconstituted in water comprises

(a) from 5 mM to 30 mM histidine,
(b) from 0 1M to 0 3M glycine,
(c) from 0 5 to 2 percent sucrose, and
(d) from 0 001 to 0 05 percent polysorbate
15 The method as claimed in claim 14, wherein prior to the addition of the sodium chloride
solution, the pharmaceutical formulation if reconstituted in water further comprises
(e) from 50 lU/mL to 2000 IU/mL of Factor IX

ABSTRACT

TITLE: METHOD FOR PREPARING A FACTOR IX FORMULATION
A method for preparing a Factor IX formulation for intravenous injection, the method
comprising adding a 25 mM to 150 mM sodium chloride solution to a lyophilized Factor IX
formulation thereby resulting in a formulation prepared for intravenous injection, wherein the
lyophilized formulation, if reconstituted in water, does not contain more man 5 mM of an
ionizing salt, and, wherein the prepared formulation
is isotonic with respect to plasma and has an osmolality that is from 270 mOsm/L to 330
mOsm/L or
is slightly hypotonic with respect to plasma and has an osmolality that is from 220 mOsm/L
to 270 mOsm/L or,
is slightly hypertonic with respect to plasma, and has an osmolality that is from 330 mOsm/L
to 600 mOsm/L and,
wherein the prepared formulation has a sufficient ionic strength to prevent erythrocyte
agglutination upon intravenous injection