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Impact of Freezing on pH of Buffered Solutions and Consequences for Monoclonal Antibody Aggregation

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Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use-period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature-associated change in the pK(a)s, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well-studied system, shows the greatest change in pH when going from +25 to -30 degrees C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to -30 degrees C, whereas Tris-hydrochloride had an approximately 1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0 degrees C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze-thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation.
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Impact of Freezing on pH of Buffered Solutions and Consequences
for Monoclonal Antibody Aggregation
Parag Kolhe, Elizabeth Amend, and Satish K. Singh
Biotherapeutics Pharmaceutical Sciences, Pfizer Global Research and Development, Pfizer Inc, Chesterfield, MO 63017
DOI 10.1002/btpr.377
Published online December 28, 2009 in Wiley InterScience (www.interscience.wiley.com).
Freezing of biologic drug substance at large scale is an important unit operation that ena-
bles manufacturing flexibility and increased use-period for the material. Stability of the bio-
logic in frozen solutions is associated with a number of issues including potentially
destabilizing pH changes. The pH changes arise from temperature-associated change in the
pK
a
s, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes
for most of the common protein formulation buffers in the frozen state have not been system-
atically measured. Sodium phosphate buffer, a well-studied system, shows the greatest
change in pH when going from þ25 to 30C. Among the other buffers, histidine hydro-
chloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change
(increase) was observed over the temperature range from þ25 to 30C, whereas Tris-
hydrochloride had an 1.2 pH unit increase. In general, a steady increase in pH was
observed for all these buffers once cooled below 0C. A formulated IgG2 monoclonal anti-
body in histidine buffer with added trehalose showed the same pH behavior as the buffer
itself. This antibody in various formulations was subject to freeze/thaw cycling representing
a wide process (phase transition) time range, reflective of practical situations. Measurement
of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was
not a factor for aggregate formation in this case, which instead is governed by the presence
or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant,
longer phase transition times lead to higher aggregation. V
V
C2009 American Institute of
Chemical Engineers Biotechnol. Prog., 26: 727–733, 2010
Keywords: formulation buffers, proteins, pH change during freezing, monoclonal antibody,
aggregation, freeze–thaw cycling
Introduction
Freezing and thawing of large volumes of bulk protein solu-
tions has become an important step in the manufacture of bio-
therapeutics because of the flexibility it affords in terms of
maximizing productivity and enabling drug product logistics in
alignment with market demands. Storing bulk protein drug
substance for periods of time in the frozen state enables a
decoupling of the drug substance production from that of drug
product. Freezing of drug substance also offers distinctive
advantages, such as minimization of risk of microbial growth,
increased product stability with extended shelf life, elimination
of agitation and foaming during transportation, and increased
flexibility during manufacturing.
1,2
However, freeze and thaw
processing also presents fundamental scientific and engineering
challengesespeciallywhencarriedoutinlarge-scalesystems,
with the freeze step being especially complicated.
3,4
Freezing
can induce complex physical and chemical changes in the sol-
vent/solute conditions, resulting in denaturation of proteins
with the possibility of generation of irreversible aggregates
over time. All the changes arise because as ice crystals grow
and remove water from solution, they exclude all solute mole-
cules including the protein, leading to zones where protein can
be found at high concentration in the presence of other cryo-
concentrated solutes (buffer species and other excipients).
3–5
Although a number of phenomena determine the overall suc-
cess of the freezing process (such as ice interface adsorption
and unfolding, dessication, vitrification, storage temperature
vis-a-vis glass transition temperature), we will restrict the dis-
cussion in this article to the effect of the freezing on the pH of
the buffer making up the protein formulation.
It is well known that pH of conventional buffers can
change with temperature. Temperature dependence of the
ionization enthalpy determines the sensitivity, but for most
practical purposes computed/tabulated dpK
a
/dT values are
used.
6–11
However, these values are only relevant to the liq-
uid state. Impact of freezing on pH has been commonly
exemplified with phosphate buffer made of sodium salts
where reaching the solubility limit of the dibasic salt leads
to a drastic drop in the pH from 7 to around 4 between about
0.5 and about 9.9C (dependent on strength of buffer,
cooling rate, etc.).
12–15
The potassium salts of phosphate on
the other hand do not have this limitation, though a minor
increase in pH due to crystallization of the monobasic salt at
6C has been shown.
16
Larsen
17
has provided frozen-
state pH data on some other buffers of interest.
17
Cryocon-
centration of solutes (excipients) during freezing is expected
Correspondence concerning this article should be addressed to S. K.
Singh at satish.singh@pfizer.com
V
V
C2009 American Institute of Chemical Engineers 727
to impact the structure of the protein solution through the
change in pH and ionic strength. Denaturation caused by
structural perturbations due to pH change has been shown
for enzymatic systems, as measured by loss of activity.
18–20
Multimeric enzymes seem to be particularly susceptible
because of loss of active-site conformation. Aggregation of
antibodies (as measured by size exclusion chromatography)
frozen in phosphate buffer systems containing salts has also
been reported.
21,22
We have therefore measured the pH–tem-
perature profile in detail for some buffers of interest for anti-
body formulations, as well as in a formulated IgG2
monoclonal antibody (mAb) solution, going down to temper-
atures of practical interest in frozen storage of protein solu-
tions. The measurements have been primarily performed in
the freezing mode. This is the most relevant part of a freeze/
thaw process when considering long-term storage of proteins
in the frozen state, because impact on protein over storage
time will be determined by the conditions generated through
freezing. The storage temperature is also critical to the long-
term impact but is outside the scope of this work.
3,4
The
thaw process, if carried out improperly (e.g., without gentle
agitation), could also lead to degradation but generally has
little long-term impact because the process brings the protein
into its stable solution state.
Among the parameters considered important are rate of
processing (cooling and heating rate) and composition. The
impact of ‘‘rates’’ as presented in the literature does not pro-
vide any clear guidance as the terms ‘‘fast’’ and ‘‘slow’’ are
specific to the study, and rates (if) reported in the literature
vary widely.
5
Furthermore, the use of minimal volumes makes
the ‘‘process’’ aspect of literature studies difficult to extrapolate
to bulk protein solutions. ‘‘Rate’’ as often used in the literature
refers to the drop in temperature over time. However, the
more important ‘‘rate’’ parameter relates to the time required
for the solution to actually freeze or thaw, that is, the actual
time spent in phase transition between onset of nucleation and
completion of ice formation or vice versa. This aspect is con-
trolled in this work by varying the volume of sample and the
freezer temperature, thus providing a range of times over
which the solution freezes and thaws. We have also subjected
the IgG2 antibody to freeze–thaw cycles under similarly vary-
ing rates, to show that buffer-induced pH change is not the
factor that impacts aggregation on freezing.
Material and Methods
Materials
InLab
V
R
Cool pH probe from which contains FRISCOLYT-
B
V
R
as a reference electrode was procured from Mettler-Tol-
edo. Kaye validator from GE was used to monitor the pro-
cess as well as buffer solution temperature. A Lauda Proline
RS485 benchtop circulating bath (silicone HTF) was used to
control and program the temperature ramps. The chemicals
used for preparation of buffers were of analytical grade and
were used as received.
The IgG2 mAb solution was studied in three formulations:
i. at 20 mg/mL in a 20 mM histidine HCl buffer, pH 5.5
with 84 mg/mL trehalose dihydrate, and 0.2 mg/mL polysor-
bate 80
ii. at 20 mg/mL in a 20 mM histidine HCl buffer, pH
5.5, and
iii. at 5 mg/mL in a 20 mM sodium acetate buffer, pH
5.5 with 8.2 mg/mL NaCl, and 0.2 mg/mL polysorbate 80.
pH measurement
The setup used for pH measurement was designed to range
between þ25 and 30C. Approximately 25 mL of buffer so-
lution was added to a centrifuge tube. The InLab
V
R
Cool pH
probe containing FRISCOLYT-B
V
R
as a reference electrode
was immersed into the solution and the tube covered with par-
afilm. The pH was measured by Navi pH meter (model F-55).
The circulating bath was used to program the temperature
change. Kaye validator probes were immersed in the bath as
well as in the buffer solution so that the temperature of the
bath along with buffer solution was monitored. Figure 1 shows
a typical temperature profile obtained during the measure-
ments. The pH probe was calibrated with pH standard solu-
tions of 4 and 7 before every experiment. During cooling, the
pH was allowed to come to a steady value before a reading
was taken. At least 5 min was allowed for the pH meter read-
ing at every temperature except for temperatures below
20C, where at least 2 min was allowed as the observed pH
changes were not significant and settled rapidly.
Freeze/thaw cycling
Freeze/thaw cycling studies on the IgG2 mAb were carried
out by freezing in 20, 40, or 70C freezers and thawing
at room temperature with gentle agitation. Impact of rate of
freezing was studied by varying the fill volume as well as
freezer temperature. Formulation (i) was tested as 1 mL fill
in 1-mL vials, and 50 mL fill in 50-mL glass vials. Formula-
tions (ii) and (iii) were tested as 50 mL fills in the 50-mL
vials at 40 or 70C only.
Test vials for the study were loaded in a tray at defined
positions, some distance apart from each other to avoid ther-
mal interaction between them. The tray was placed in the
freezers in a defined location. Temperature profiles of solution
in the vials were recorded separately using the Kaye validator.
Vials placed at different locations in the tray had very similar
profiles. Use of 1- and 50-mL vials in different freezers led to
widely different freezing and thawing times, measured as the
time required for the phase transition to complete. The phase
transitiontimesrangedbetween55min(1mLin70C
freezer) and 830 min (50 mL in 20C freezer).
Size exclusion chromatography
Size exclusion chromatography (SE-HPLC) was used to
detect soluble aggregate levels in the mAb samples. The
Figure 1. Typical temperature profile during the determina-
tion of pH as a function of temperature.
728 Biotechnol. Prog., 2010, Vol. 26, No. 3
column used for this analysis was TSK3000SWXL (300 mm
7.8 mm) in a 50 mM sodium phosphate pH 7.0 with 600
mM sodium chloride running buffer at 1.0 mL/min over 20
min. Detection was by UV absorbance at 214 nm.
Results
pH of buffers during freezing
Changes in pH of commonly used formulation buffers
were monitored as a function of temperature from þ25 to
30C. Various common formulation buffers used in this
study are summarized in Table 1 along with the difference in
pH observed at þ25, 0, and 30C, dpK
a
/dT values (liquid
state). The data are shown in Figures 2–8 as plots of pH vs.
temperature. To aid in the understanding of the results, we
have also plotted liquid-state pK
a
values (at the specified ionic
strength, I) from the literature (Goldberg et al.
6
)aswellascal-
culated pHs using literature reported dpK
a
/dT. Note that the
pH measured in this situation is that of the cryoconcentrating
solute phase excluded by the formation of ice. [As part of this
study, a 20 mM phosphate buffer with 350 mM arginine, pH
7.5 was also examined. On cooling between þ25 and 10C,
the pH fluctuated about 0.2 units. Below this temperature, the
pH started fluctuating wildly between 2and11. No cause
for this could be ascertained, and the results of this buffer are
not presented here.]
Phosphate Buffer. To ensure that the experimental system
and pH probe provided reliable results, we started by examin-
ing a 50 mM sodium phosphate buffer at pH 7. The pH of
this buffer as a function of temperature is shown in Figure 2.
It can be seen that for 50 mM sodium phosphate buffer of pH
7, no significant changes in the pH was observed down to
0C, consistent with its pK
a
behavior. A precipitous drop in
pH was observed as the temperature was decreased below 0C
because of saturation of the dibasic salt. At 15C, a pH drop
of 3.1 units was measured compared with that at 0C. The pH
gradually decreased as the temperature was lowered further,
and a final pH value of 3.4 was measured at 30Cindicating
a total pH drop of 3.6 units. The slower decrease after
10C is consistent with completion of the eutectic crystal-
lization of Na
2
HPO
4
at this temperature, after which a single
saturated phase freezes. These observations are consistent with
those reported previously by several authors, confirming the
suitability of our system, although results down to 30C
have not been reported previously.
13–15,19,23
Athawofthefro-
zen buffer was also performed. As shown in the Figure 2, the
pH after thaw returned to the original value at 25Cwitha
slight hysteresis.
Acetate Buffer. Figure 3 shows the changes in pH
observed for 20 mM sodium acetate buffer, pH 5.6 at
þ25C. Consistent with observations that carboxylic acids
have dpK
a
/dT values close to zero, no change is seen down
to 0C. Below 0C, a linear increase in the pH value was
observed as a function of decreasing temperature. The final
pH observed for the sodium acetate buffer was 6.1 at
30C, an increase of 0.5 units in pH from þ25C and
Table 1. Summary of Changes in pH for Various Formulation Buffers as a Function of Temperature
Buffer Solution
pH Measured
at 25C
pH Measured
at 0C
dpK
a
/dT in Liquid
State (Literature)
pH Measured
at 30C
Lowest pH
Measured
Highest pH
Measured
Net Change
in pH (25 to
30C)
50 mM Sodium phosphate 7.00 7.02 þ0.0044 (pK
a1
), þ0.0028
(pK
a2
), 0.026 (pK
a3
)*
3.36 3.36 at 30C 7.00 at 25C 3.64
20 mM Sodium acetate 5.63 5.66 þ0.0002* 6.14 5.61 at 5C 6.14 at 30C 0.51
20 mM L-Histidine-HCl 5.37 5.86 0.022** 6.19 5.37 at 25C 6.14 at 30C 0.82
20 mM Histidine-acetate 5.52 5.97 Combined effect 6.48 5.52 at 25C 6.48 at 30C 0.96
20 mM Sodium citrate 6.16 6.49 0.0016 (pK
a2
), 0.0 (pK
a3
)* 5.93 5.69 at 10C 6.17 at 20C 0.23
20 mM Tris-HCl 7.37 7.93 0.028* 8.54 7.37 at 5C 8.54 at 30C 1.17
20 mM Sodium succinate 5.55 5.60 0.0018 (pK
a1
), 0.0 (pK
a2
)* 5.85 5.49 at 10C 5.85 at 30C 0.3
*All values from Stoll and Blanchard.
11
** Calculated from pK
a2
values reported for histidine in Perrin.
9
Note that dpK
a
/dT for imidazole is 0.020.
11
Figure 2. Change in pH as a function of temperature for so-
dium phosphate buffer when the temperature was
decreased from 125 to 2308C and increased from
230 to 1258C.
(hindicates pK
a
values from Goldberg et al.
6
).
Figure 3. Change in pH as a function of temperature for ace-
tate buffer when the temperature was decreased
from 125 to 2308C.
(hindicates pK
a
values from Goldberg et al.
6
; calculated pH
based on values of dpK
a
/dT in Table 1).
Biotechnol. Prog., 2010, Vol. 26, No. 3 729
from 0C. The literature pK
a
values (I¼0) for dissociation
of acetic acid at various temperatures are also plotted in the
Figure 3 for reference and provides confirmation for the
results because the pH change (liquid state) parallels the pK
a
plot. Finally, the data from Larsen
17
are also plotted. There
is some general agreement in the data below 0C with
Larsen
17
reporting a 0.2 unit increase, whereas our measure-
ments give a 0.26 unit increase over the same temperature
range. Clearly, no solubility limits are reached that would
lead to abrupt changes in pH. Acetate buffer appears to have
low crystallization potential when compared with other buf-
fers.
24
Cooling below the eutectic temperature of sodium ac-
etate (18C) leads to a further steady increase in pH.
L-Histidine (Hydrochloride) Buffer. Temperature–pH pro-
files of 20 mM histidine HCl buffer are shown in Figure 4.
A significant linear increase in the pH was observed down to
0C, consistent with general behavior of amines, considering
the imidazole group as an aliphatic amine. There is a reduc-
tion in the rate of increase of pH below 0C, but as the tem-
perature is lowered to 30C, the increase in pH continues.
The final pH observed for the histidine HCl buffer was 6.2
at 30C, an increase of 0.8 units in pH from þ25C, and
0.3 units from 0C. Calculated values for pH as a function
of temperature in the liquid state showed a close agreement
with the observed pH values. Literature pK
a2
values (I¼0
and 100 mM) (for the basic nitrogen on the imidazole group)
as a function of temperature are also plotted and show that
the measured pH values run in parallel. The temperature–
pK
a2
values for histidine show a significant effect of ionic
strength. The pH of this buffer was also measured in the
thaw mode and shows good agreement with the freeze curve.
The slight hysteresis is likely due to the effect of cryocon-
centration of buffer, that is, effectively a higher buffer
strength with a higher pK
a
at the same temperature. Histidine
remains amorphous during freezing, but can crystallize out
on thawing for pH ranges outside 5.5–6.5.
25,26
The lack of
abrupt changes in pH in Figure 4 is in agreement with the
proposition that solubility limits were not reached and/or
crystallization did not occur in the system investigated.
Histidine Acetate Buffer Solution. Under circumstances
where chloride ions are not acceptable in the formulation, it
may be replaced by acetate ions (see e.g., Chen et al.
27
). A
combination buffer of histidine (20 mM) and acetate (13.82
mM) was prepared at pH 5.5 and monitored as a function of
temperature (Figure 5). The change in pH when the tempera-
ture is lowered from 25 to 0C follows the trend for histi-
dine, as no contribution to the change is expected from the
acetate component. Further decrease in temperature below
0C leads to the pH continuing to increase. The final pH
observed for the histidine acetate buffer was 6.48 at 30C,
an increase of 1 unit in pH from þ25C, and 0.45 units
from 0C. Although not exact, the change of pH in this
range can also be accounted for by the changes of pK
a
of
histidine and acetate, added together in the appropriate molar
ratio.
Tris-HCl Buffer Solution. Tris-HCl as a primary amine
shows significant changes in pH as a function of temperature
(Figure 6). The initial pH for the buffer at þ25C was 7.37.
When the temperature was decreased, an increase in pH val-
ues was observed similar to the behavior observed for histi-
dine as well as histidine-acetate buffers. The change in pH
parallels the change in pK
a
and interestingly continues with
the same slope into the frozen state also. The final pH
observed for the Tris-HCl buffer was 8.54 at 30C, an
increase of 1.2 units in pH from þ25C, and 0.6 units
Figure 4. Change in pH as a function of temperature for histi-
dine buffer when the temperature was decreased
from 125 to 2308C.
(h,nindicate pK
a
values from Goldberg et al.
6
; calculated pH
based on values of dpK
a
/dT in Table 1).
Figure 5. Change in pH as a function of temperature for histi-
dine acetate buffer when the temperature was
decreased from 125 to 2308C.
Figure 6. Change in pH as a function of temperature for Tris-
HCl buffer when the temperature was decreased
from 125 to 2308C.
(hindicates pK
a
values from Goldberg et al.
6
; calculated pH
based on values of dpK
a
/dT in Table 1).
730 Biotechnol. Prog., 2010, Vol. 26, No. 3
from 0C. Data from Larsen
17
are in general agreement with
our results, showing a slight tendency for increase in pH
below 0C, but the measured values are different. Tris(hy-
droxyl methyl amino methane) (CH
2
OH)
3
C(NH
2
) has a
eutectic at 5.3C and therefore has the potential to reach
its solubility limit. However, the pH in the frozen state is
trending toward basic, implying that no solubility limit is
reached in our experimental system.
Citrate Buffer. Citrate buffers, as a (tri)carboxylic acid,
show a very weak dependence of pK
a
on temperature in the
solution state, as also seen by the literature pK
a2
and pK
a3
values plotted in Figure 7. We monitored the changes in pH
as a function of temperature for two citrate molarities (5 and
20 mM) for three different pH values of 5.5, 6.0, and 6.5
(Figure 7). The main change occurs between 0 and 10C,
probably due to precipitation of sodium citrate. After this
process is complete below 10C, a small increase in pH is
seen. There is no significant impact of buffer strength in the
range studied. Trends in the data from Larsen
17
are again in
general agreement with the results of this study. The results
are also consistent with that reported by Orii and Morita,
where no significant changes in sodium citrate buffer at pH
5.5 were observed by using pH indicators.
28
Succinate Buffer. Succinic acid, as a (di)carboxylic acid,
shows little change in pK
a
with temperature in liquid state
Stoll and Blanchard report a dpK
a1
/dT ¼0.0018 and
dpK
a2
/dT ¼0. Our measurements are in agreement (Figure
8) for the temperature range above 0C. A small acidic shift
(0.1 units) occurs on cooling just below this point, but the
pH starts to increase below 15C. Lam et al. hypothesized
the crystallization of sodium succinate salt (at 30C) lead-
ing to an acidic shift in pH, as a cause for the poor stability
of Ifn-cwhen lyophilized in succinate buffer.
29
Crystalliza-
tion temperature and extent of crystallization was dependent
on pH
30
and would be expected to occur around 25C for
a buffer of pH 5.5. However, in the previous reports, crystal-
lization has been only observed during heating step and not
during cooling.
29,30
This suggests that our observation of the
slight increase in pH during cooling is likely not related to a
crystallization event, but simply a general trend of increase
in pH in the frozen state as the temperature drops.
Histidine Formulation Buffer Containing mAb. Tempera-
ture–pH profiles of histidine formulation (i) [containing 20
mg/mL mAb along with trehalose and polysorbate 80] are
shown in Figure 4 and agree well with the profiles of the
histidine buffer itself. This confirms that the buffer in this
case is a good representative of the formulation behavior
during freezing and thawing.
Freeze/thaw cycling
Disaccharides such as trehalose or sucrose stabilize the
protein by preferential exclusion mechanism and are added
to protein formulations as cryoprotectants.
31,32
Sodium chlo-
ride generally does not serve as a good cryoprotectant
because of its tendency to form a eutectic and crystallize out
of solution during freezing.
3–5
This study tried to separate
the impact of presence/absence of these excipients from any
potential effect of pH changes on protein stability during
freeze/thaw cycling. Nonionic surfactants such as polysor-
bate 80 also protect against freezing-induced aggregation by
preventing or reducing unfolding of the protein at the ice–
liquid interface.
33
However, this study was not designed to
explicitly examine the effect of this excipient.
Size exclusion chromatography results on the mAb solu-
tions after a number of freeze/thaw cycles are shown in Fig-
ure 9. Lack of change in formulation (i) shows that for this
mAb, pH change on freezing in itself has no impact on solu-
ble aggregation levels because of freeze/thaw processing.
However, presence of a cryoprotectant is critical as the data
for formulation (ii) (Figure 9) show that lack of a suitable
cryoprotectant (trehalose) leads to significant aggregate for-
mation. In agreement with this, the acetate formulation con-
taining NaCl (iii) also shows significant aggregation. The pH
change in the frozen state for acetate buffer is of the same
order as in the histidine buffer. However, NaCl crystallizes
on freezing and does not function as a cryoprotectant. In this
system, the presence of surfactant seems to have little pro-
tective impact because formulation (ii) without surfactant
(and no other additive) shows lower overall aggregation lev-
els than formulation (iii) that does contain this surfactant
(but also contains NaCl). Finally, the data in Figure 9 show
Figure 7. Change in pH as a function of temperature for ci-
trate buffers of various pH (5.5, 6.0, and 6.5) and
molarity when the temperature was decreased from
125 to 2308C.
(hindicates pK
a
values from Goldberg et al.
6
).
Figure 8. Change in pH as a function of temperature for succi-
nate buffer when the temperature was decreased
form 25 to 2308C.
(hindicates pKa values from Goldberg et al.
6
).
Biotechnol. Prog., 2010, Vol. 26, No. 3 731
that the mAb, although susceptible to aggregation, is pro-
tected by a well-designed formulation (i) when subject to
multiple freeze/thaws over a wide range of process condi-
tions. The freezing (phase transition) time ranged between
55 min (1-mL in 70C freezer) and 830 min (50-mL in
20C freezer) (not shown), and it is this transition period
that is most detrimental to the protein undergoing freeze/
thaw.
18
Formulations (ii) and (iii) in Figure 9 show slightly
higher aggregation when frozen at 40C instead of 70C
probably reflecting impact of time of freezing in the system
without cryoprotectants, although for formulation (iii), the
levels after 10 cycles become similar. Note that in this study,
the impact of freeze/thaw cycles was only assessed by meas-
uring the level of soluble aggregates by SE-HPLC. In fol-
low-on studies, we have seen that such treatment also
generates proteinaceous subvisible (2lm size) particles,
with a much larger number of particles arising in formula-
tions that contain NaCl compared with trehalose (unpub-
lished data). This is in agreement with the observations on
soluble aggregate made in this study. The SE-HPLC method
used here will not detect these particles as they are filtered
out by the column. Further discussion of the impact of such
subvisible particles is beyond the scope of this study, but it
may suffice to note that from a bulk drug substance perspec-
tive, filtration of the bulk solution through 0.22-lm filters
before filling into vials is standard practice in the industry.
Discussion
The pH of a number of pharmaceutically relevant buffers
has been measured in the frozen state down to 30C. The
measurements have been primarily performed in the cooling/
freezing mode. Results in the liquid state down to 0C agree
with literature. Carboxylic acids (acetic, succinic, and citric)
and inorganic acids (phosphoric) have low dpK
a
/dT, while
primary amines (tris) are significantly impacted. Histidine, a
popular buffer component for proteins especially mAbs, also
has a large dpK
a
/dT coefficient because of its imidazole moi-
ety, an aliphatic amine. Once frozen, the pH behavior is
generally determined by solubility of the least-soluble com-
ponents of the buffer. Although the behavior of a sodium
phosphate buffer is well known, information on the other
buffers is very limited. In the frozen state, apart from the so-
dium phosphate buffer, the other buffers studied do not show
any significant pH change driven by solubility limitation of
any component. These buffers show nearly linear increases
in pH ranging between 0.4 and 0.8 units from the pH at 0C,
when cooled down to 30C. This seems to be a general
behavior likely not related to any solubility limitations but
driven by activity or viscosity effects.
34
Combining buffer
species such as histidine and acetate leads to an additive
effect on the temperature–pH profile. A similar observation
has been recently utilized in the preparation of temperature-
resistant pH buffers where buffers with positive and negative
temperature coefficients have been combined,
35
although a
number of the biological buffers (e.g., MOPS, MES, HEPES,
and BisTrisPropane) required to create these systems have
not been used in parenteral pharmaceutical formulations. Ex-
amination of the dpK
a
/dT tables in Stoll and Blanchard
11
shows that apart from phosphate (pK
a1
), the majority of buf-
fers, including pharmaceutically relevant ones, have negative
or zero temperature coefficients. It is only the biological buf-
fers of the type listed earlier that have the positive tempera-
ture coefficients.
Information about pH–temperature effect can be used to
design formulation development studies and evaluate poten-
tial impact of freezing-induced pH change vs. other effects
of freezing (e.g., ice interfacial adsorption, cryoconcentra-
tion, and phase separation) on the stability of the protein.
Freeze/thaw studies on an IgG2 mAb formulated in histidine
or acetate buffers show that pH change due to buffer can be
ruled out as a factor in the aggregation behavior. Instead, the
absence of cryoprotectant is the key determinant of freeze/
thaw aggregation. This mAb has optimum liquid-state stabil-
ity at pH 5.5 and shows minor increases in aggregation over
time at pH 6.0 (data not shown). Although the two systems
are not directly comparable, the observation of lack of
impact of change of pH in frozen state in this work is in
general agreement with the observation of slow increase in
aggregation at pH 6.0 when in the liquid state.
Although protein molecules differ in their intrinsic aggre-
gation propensity,
36
it is possible to deconvolute the mecha-
nism of freeze/thaw-induced aggregation from the pH-
induced destabilization. Trehalose as a noncrystallizing
additive protects through the preferential exclusion mecha-
nism and/or by reducing the cold denaturation temperature
(extrapolating from Tang and Pikal
37
), during the (phase)
transition period when the system is cryoconcentrating but is
not completely kinetically immobilized. It is in this transition
period that the protein can be denatured at the ice interface
or through any pH-change induced stress.
37
The mAb in the
trehalose formulation shows the lack of impact of a wide
range of freeze process time, while the absence of a cryopro-
tectant enhances the impact of process time. On the other
hand, increasing concentrations of NaCl during freezing can
also cause the protein to salt-out and probably leads to the
aggregation seen after freeze/thaw. Furthermore, NaCl crys-
tallizes out at its eutectic point (21.2C, 22 wt % NaCl
or 3.7 M).
38
Crystallized excipients are known to have no
cryoprotective (or lyoprotective) effect (see e.g., Mi et al.
39
).
Conclusions
We have measured the changes in pH for commonly used
protein formulation buffers as the buffer is cooled from þ25
Figure 9. Soluble aggregate levels as measured by size exclu-
sion chromatography for mAb solutions in different
formulation buffers when subject to various freeze–
thaw cycles.
732 Biotechnol. Prog., 2010, Vol. 26, No. 3
to 30C. Apart from the well-known behavior of sodium
phosphate buffer, the other buffers tested here show small
(\1 unit) pH change when cooled below 0C. Dramatic
changes due to solubility limitations were not detected. Com-
bining buffer species was observed to lead to an additive
effect on the temperature–pH profile. The information can be
used to design formulation development studies and evaluate
potential impact of freezing-induced pH change on the sta-
bility of the protein. Studies with an IgG2 mAb showed that
the buffer-induced pH change could be ruled out as a factor
for aggregate formation in this case, which instead was gov-
erned by the presence or absence of noncrystallizing exci-
pients. In the absence of a cryoprotectant, longer phase
transition times lead to higher aggregation.
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Biotechnol. Prog., 2010, Vol. 26, No. 3 733
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Twelve new or little used hydrogen ion buffers covering the range pKa = 6.15-8.35 have been prepared and tested. Ten are zwitterionic amino acids, either N-substituted taurines or N-substituted glycines, and two are cationic primary aliphatic amines. All of the zwitterionic buffers are better than conventional buffers in the Hill reaction and in the phosphorylation-coupled oxidation of succinate by bean mitochondria. Two of the zwitterions, N-tris(hydroxymethyl)-methylaminoethanesulfonic acid and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, give particularly active and stable mitochondrial preparations. These two also give higher rates of protein synthesis in cell-free bacterial preparations than do tris(hydroxymethyl)aminomethane (Tris) or phosphate buffers.
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Buffers are commonly used to control pH when freeze-drying pharmaceuticals and biologicals. This paper reviews lyophilization-related properties of pharmaceutical buffers such as buffer crystallization, pH changes, and collapse behavior during freezing. It is recommended that a suitable buffer for freeze-drying should be non-volatile, have a high collapse temperature (Tg'), and remain amorphous during freeze-drying. Citrate buffer appears to be a good choice for lyophilized formulations that are prepared at acidic or near neutral pH. Frozen solutions of citrate buffer have a low crystallization potential, relatively high collapse temperature, and minimal pH changes during freezing. Alkaline buffers, however, have been studied to a lesser degree, and no obvious buffer candidates can be recommended for lyophilized formulations at basic pH values. In addition, the paper highlights a need to better understand the impact of amorphous solutes on buffer crystallization and pH changes during lyophilization.