Dry Powder Therapeutic mAb Formulations with … · Dry Powder Therapeutic mAb Formulations with...
1
Dry Powder Therapeutic mAb Formulations with Enhanced Temperature Stability F. McNamee 1 , W. Gebbie 1 , K. Davidson 1 , J. Partridge 1 , J. Vos 1 , J. Abate 2 , C. Kirchhoff 2 , B. D. Moore 1 XstalBio Ltd, Thomson Building, University Avenue, Glasgow, G12 8QQ, U.K. 1 Pfizer Global Research & Development, St. Louis, MO, USA. 2 www.xstalbio.com email address: [email protected] Conclusion Human monoclonal antibodies can be readily formulated using PCMC technology by incorporating precipitation stabilizing additives (PSA). PCMC coprecipitation leads to finely-divided dry powders, which can be rapidly reconstituted back into aqueous, to release the monoclonal antibody in monomeric form. Such PCMC mAb dry powders are attractive as a platform for alternate delivery applications. Discussion During the PCMC process, protein molecules are exposed to a very different environment to that arising during lyophilisation or spray-drying. For molecules prone to self- association this can lead to a requirement for novel stabilising excipients. In this work we have demonstrated that a combination of glutamic acid and arginine is able to keep mAbs in a monomeric form during dehydration and precipitation using polar solvents. Lyoprotectants such as trehalose are much less effective. It is hypothesised that: • within solvent, protein association is predominately via charge-charge interactions • neutral additives such as trehalose cannot prevent this • Glu and Arg additives ion-pair with charged protein side-chains • a zwitterion-coating minimises intermolecular mAb association in dry-state • additional neutral additives act synergistically by displacing water molecules A combination of Glu and Arg has previously been reported to be useful for preventing protein precipitation in highly concentrated aqueous protein solutions with minimal reduction of specific protein-protein interactions (studied by NMR; Golovanov, A. et al., A Simple Method for Improving Protein Solubility and Long-Term Stability, JACS, 2004, 8933-8939). This observation appears contradictory to the above hypothesis. However, this can be explained by the much weaker ion-pairing in water and the importance of hydrophobic interactions for driving protein association. Recent developments of this technology have provided powders that can be rapidly reconstituted to produce very high concentration mAb solutions (>200 mg/mL), deliverable through a 27 gauge needle, with acceptable glide force and osmolality. Bioactivity of PFCP PCMC coprecipitation preserves the activity of the mAb. The bioactivity of the PFCP samples was tested in a PFCP specific ELISA. From the results it is clear that bioactivity has not been compromised by the PCMC coprecipitation process. Furthermore the protein loading measured is approximately equivalent to the theoretical composition, demonstrating that protein is not lost in the coprecipitation process, but is fully immobilized on the surface of the microcrystal. Sample Theoretical Protein Loading (%w/w) Measured Protein Loading (%w/w) % Activity PFCP1_56_1 16.8 15.6 92 PFCP1_56_2 17.2 16.8 95 PFCP1_56_3 32.7 30.0 108 PFCP1_56_4 32.7 28.5 109 PFCP1_56_5 26.6 26.1 107 PFCP1_56_6 26.6 23.4 96 Introduction Protein Coated Micro Crystal (PCMC) technology was used to process a human therapeutic monoclonal antibody into dry powder formulations, which were studied under accelerated stress conditions. Changes in protein integrity on reconstitution were measured by size exclusion chromatography and turbidity measurements. The effect of glutamic acid (Glu), L-arginine (Arg) and trehalose as precipitation stabilising additives was investigated. PCMC Technology PCMC are produced by coprecipitation of biomolecule and coprecipitant into GRAS solvent. The PCMC are formed by a rapid, self-assembly process, whereby the coprecipitant core (blue cubes) forms a support core and the biomolecule (yellow spheres) is immobilized on this crystal surface. solvent coprecipitation Abstract XstalBio have developed platform technologies for stabilizing a wide range of biotherapeutics within a dry powder format. By using a rapid isothermal precipitation process, dry powder products can be prepared for a range of administration strategies including inhalation, high concentration subcutaneous injection (>200 mg/mL) and sustained release. The aim of this study was to optimise dry powder formulations of a human monoclonal antibody to achieve a highly extended shelf-life. Materials & Methods Monoclonal human antibody, herein termed PFCP1, was obtained from Pfizer Inc, Chesterfield, St. Louis. PFCP1 dry powders were prepared by coprecipitation of an aqueous mixture containing histidine buffered antibody, concentrated glycine coprecipitant and PSA (L-arginine & L-glutamic acid), into either propan-2-ol or 2-methyl-1-propanol. An accelerated stress study was then carried out in which PFCP1 dry powders were stored at 40ºC at uncontrolled humidity for ~47 weeks. After temperature stressing, the PFCP1 dry powder was reconstituted back into buffer and monomer content was measured by size- exclusion chromatography. Results All of the stressed samples retained ≥95% monomer content. Bioactivity was tested in a PFCP1 specific ELISA; the results showed that bioactivity was also not compromised by the solvent coprecipitation process. Conclusions Human monoclonal antibodies can be readily formulated using XstalBio technology by incorporating precipitation stabilizing additives (PSA). Coprecipitation leads to finely divided dry powders, which can be rapidly reconstituted back into aqueous. Moreover, conversion to this dry format imparts excellent thermostability to the mAb. Applications to Biotherapeutics Recent developments of this technology have provided powders that can be rapidly reconstituted to produce very high concentration mAb solutions (>200 mg/mL), deliverable through a 27 gauge needle, with acceptable glide force and osmolality. Following coprecipitation, the suspension of PCMC particles was filtered and left to air dry overnight. Human mAb PCMC Formulations Monoclonal human antibody, PFCP, was obtained from Pfizer Inc, St Louis, MO. PFCP is a fully human monoclonal antibody specific for human cytotoxic T lymphocyte–associated antigen 4. PFCP PCMC were prepared by coprecipitation of an aqueous mixture of histidine buffered antibody and concentrated glycine coprecipitant into either propan-2-ol or 2-methyl-1-propanol. PCMC were prepared in the presence and absence of the precipitation protective additive pair (Glu, Arg) and with and without trehalose. aqueous mixture of concentrated coprecipitant and biomolecule dropwise addition with stirring water-miscible solvent biomolecule- coated microcrystals The ratio of active mAb to coprecipitant/PSA was varied between 17%w/w and 33 %w/w, as shown in this table (Theoretical Protein Loading (%w/w)). Theoretical Protein Loading (%w/w) Glycine (%w/w) Precipitation Stabilizing Additive (%w/w) Trehalose Dihydrate (%w/w) mAb Stock (Stored @ 4-8 º C) PFCP1_82_6 - No Additive IPA 41.1 57.0 0.0 0.0 PFCP1_82_1 - Trehalose Only IPA 32.0 44.4 0.0 22.0 PFCP1_56_1 IPA 16.8 55.7 4.5 22.3 PFCP1_56_2 IPA 17.2 56.9 13.7 11.4 PFCP1_56_3 IPA 32.7 46.4 19.4 0.0 PFCP1_56_4 2M1P 32.7 46.4 19.4 0.0 PFCP1_56_5 IPA 26.6 37.7 15.7 18.8 PFCP1_56_6 2M1P 26.6 37.7 15.7 18.8 Coprecipitation Solvent PCMC Composition (%) Sample Stability Under Accelerated Stress Conditions Monomer Content (%) Storage @ 40 oC for 47 weeks (Uncontrolled Humidity) 90 91 92 93 94 95 96 97 98 99 100 0.0 10.0 20.0 30.0 40.0 50.0 Time (Weeks) Monomer Content (%) PFCP1_56_2 PFCP1_56_3 PFCP1_56_4 PFCP1_56_5 PFCP1_56_6 Monomer Content (%) Storage @ 25oC for 13 weeks (Uncontrolled Humidity) 97.5 98 98.5 99 99.5 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (Weeks) Monomer Content (%) PFCP1_56_2 PFCP1_56_3 PFCP1_56_4 PFCP1_56_5 PFCP1_56_6 PFCP PCMC powders were stored at 25ºC in sealed vials at ambient humidity for a standard period of 13 weeks. All samples retained >98.5% monomer content. (Liquid PFCP stored @ 2-8ºC had a monomer content of 99.6% monomer.) PFCP PCMC powders were stored in sealed vials at 40ºC ambient humidity for a standard period of ~47 weeks. All samples retained ≥95% monomer content. Sample PFCP1_56_6 retained 98.5% monomer - a change of only 1.1% over 47 weeks. (Liquid PFCP stored @ 2-8ºC had a monomer content of 99.6% monomer.) Dry PCMC mAb powders incorporating Glu and Arg exhibit high stability under accelerated stress conditions. Inclusion of a further neutral additive such as trehalose enhances stability even further. Monomer Content after Coprecipitation After drying, the PFCP PCMC material was reconstituted into histidine buffer at a target protein concentration of 1 mg/mL, and monomer content was measured by size-exclusion chromatography, using a Tosoh TSKGel G3000 SW XL 7.8 mm ID x 30 cm column. These results show that the mAb remains almost exclusively as monomer when PCMC coprecipitation is undertaken with Glu and Arg present. When no additives or trehalose alone were used, significant formation of higher molecular weight species occurred. Monomer Content after Solvent Coprecipitation & Reconstitution (%) 99.7 82.7 90.87 98.87 99.31 99.49 99.5 99.52 99.64 80.0 81.0 82.0 83.0 84.0 85.0 86.0 87.0 88.0 89.0 90.0 91.0 92.0 93.0 94.0 95.0 96.0 97.0 98.0 99.0 100.0 mAb Stock (Stored @ 4- 8ºC) PFCP1_82_6 - No Additive PFCP1_82_1 - Trehalose Only PFCP1_56_1 - PSA / Trehalose / IPA PFCP1_56_2 - PSA / Trehalose / IPA PFCP1_56_3 - PSA / IPA PFCP1_56_4 - PSA / 2M1P PFCP1_56_5 - PSA / Trehalose / IPA PFCP1_56_6 - PSA / Trehalose / 2M1P Monomer Content (%) Monomer Content (%) Storage @ 25ºC for 13 weeks (Uncontrolled Humidity) Monomer Content (%) Storage @ 40ºC for 47 weeks (Uncontrolled Humidity) Monomer Content (%) Storage @ 25ºC for 13 weeks (Uncontrolled Humidity) Monomer Content (%) Storage @ 40ºC for 47 weeks (Uncontrolled Humidity)
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Transcript of Dry Powder Therapeutic mAb Formulations with … · Dry Powder Therapeutic mAb Formulations with...
Dry Powder Therapeutic mAb Formulations with Enhanced Temperature Stability
F. McNamee1, W. Gebbie1, K. Davidson1, J. Partridge1, J. Vos1, J. Abate2, C. Kirchhoff2, B. D. Moore1
XstalBio Ltd, Thomson Building, University Avenue, Glasgow, G12 8QQ, U.K.1
Pfizer Global Research & Development, St. Louis, MO, USA.2
www.xstalbio.com email address: [email protected]
ConclusionHuman monoclonal antibodies can be readily formulated using PCMC technology by
incorporating precipitation stabilizing additives (PSA). PCMC coprecipitation leads to
finely-divided dry powders, which can be rapidly reconstituted back into aqueous, to
release the monoclonal antibody in monomeric form. Such PCMC mAb dry powders are
attractive as a platform for alternate delivery applications.
Discussion
During the PCMC process, protein molecules are exposed to a very different environment
to that arising during lyophilisation or spray-drying. For molecules prone to self-
association this can lead to a requirement for novel stabilising excipients. In this work we
have demonstrated that a combination of glutamic acid and arginine is able to keep mAbs
in a monomeric form during dehydration and precipitation using polar solvents.
Lyoprotectants such as trehalose are much less effective.
It is hypothesised that:
• within solvent, protein association is predominately via charge-charge interactions
• neutral additives such as trehalose cannot prevent this
• Glu and Arg additives ion-pair with charged protein side-chains
• a zwitterion-coating minimises intermolecular mAb association in dry-state
• additional neutral additives act synergistically by displacing water molecules
A combination of Glu and Arg has previously been reported to be useful for preventing
protein precipitation in highly concentrated aqueous protein solutions with minimal
reduction of specific protein-protein interactions (studied by NMR; Golovanov, A. et al., A
Simple Method for Improving Protein Solubility and Long-Term Stability, JACS, 2004,
8933-8939). This observation appears contradictory to the above hypothesis. However,
this can be explained by the much weaker ion-pairing in water and the importance of
hydrophobic interactions for driving protein association.
Recent developments of this technology have provided powders that can be rapidly
reconstituted to produce very high concentration mAb solutions (>200 mg/mL),
deliverable through a 27 gauge needle, with acceptable glide force and osmolality.
Bioactivity of PFCP
PCMC coprecipitation preserves the activity of the mAb. The bioactivity of the PFCP
samples was tested in a PFCP specific ELISA.
From the results it is clear that bioactivity has not been compromised by the PCMC
coprecipitation process. Furthermore the protein loading measured is
approximately equivalent to the theoretical composition, demonstrating that protein
is not lost in the coprecipitation process, but is fully immobilized on the surface of
the microcrystal.
Sample Theoretical Protein
Loading (%w/w)
Measured Protein
Loading (%w/w)% Activity
PFCP1_56_1 16.8 15.6 92
PFCP1_56_2 17.2 16.8 95
PFCP1_56_3 32.7 30.0 108
PFCP1_56_4 32.7 28.5 109
PFCP1_56_5 26.6 26.1 107
PFCP1_56_6 26.6 23.4 96
IntroductionProtein Coated Micro Crystal (PCMC) technology was used to process a human
therapeutic monoclonal antibody into dry powder formulations, which were studied
under accelerated stress conditions. Changes in protein integrity on reconstitution
were measured by size exclusion chromatography and turbidity measurements. The
effect of glutamic acid (Glu), L-arginine (Arg) and trehalose as precipitation
stabilising additives was investigated.
PCMC Technology
PCMC are produced by coprecipitation of
biomolecule and coprecipitant into GRAS
solvent. The PCMC are formed by a rapid,
self-assembly process, whereby the
coprecipitant core (blue cubes) forms a
support core and the biomolecule (yellow
spheres) is immobilized on this crystal
surface.
solvent
coprecipitation
AbstractXstalBio have developed platform technologies for stabilizing a wide range of biotherapeuticswithin a dry powder format. By using a rapid isothermal precipitation process, dry powder products can be prepared for a range of administration strategies including inhalation, high concentration subcutaneous injection (>200 mg/mL) and sustained release.
The aim of this study was to optimise dry powder formulations of a human monoclonal antibody to achieve a highly extended shelf-life.
Materials & Methods
Monoclonal human antibody, herein termed PFCP1, was obtained from Pfizer Inc, Chesterfield, St. Louis. PFCP1 dry powders were prepared by coprecipitation of an aqueous mixture containing histidine buffered antibody, concentrated glycine coprecipitant and PSA (L-arginine & L-glutamic acid), into either propan-2-ol or 2-methyl-1-propanol.
An accelerated stress study was then carried out in which PFCP1 dry powders were stored at 40ºC at uncontrolled humidity for ~47 weeks. After temperature stressing, the PFCP1 dry powder was reconstituted back into buffer and monomer content was measured by size-exclusion chromatography.
Results
All of the stressed samples retained ≥95% monomer content. Bioactivity was tested in a PFCP1 specific ELISA; the results showed that bioactivity was also not compromised by the solvent coprecipitation process.
Conclusions
Human monoclonal antibodies can be readily formulated using XstalBio technology by incorporating precipitation stabilizing additives (PSA). Coprecipitation leads to finely divided dry powders, which can be rapidly reconstituted back into aqueous. Moreover, conversion to this dry format imparts excellent thermostability to the mAb.
Applications to Biotherapeutics
Recent developments of this technology have provided powders that can be rapidly reconstituted to produce very high concentration mAb solutions (>200 mg/mL), deliverable through a 27 gauge needle, with acceptable glide force and osmolality.
Following coprecipitation, the suspension of PCMC particles was filtered and left to
air dry overnight.
Human mAb PCMC Formulations
Monoclonal human antibody, PFCP, was
obtained from Pfizer Inc, St Louis, MO.
PFCP is a fully human monoclonal
antibody specific for human cytotoxic T
lymphocyte–associated antigen 4.
PFCP PCMC were prepared by
coprecipitation of an aqueous mixture of
histidine buffered antibody and
concentrated glycine coprecipitant into
either propan-2-ol or 2-methyl-1-propanol.
PCMC were prepared in the presence and
absence of the precipitation protective
additive pair (Glu, Arg) and with and
without trehalose.
aqueous mixture
of concentrated
coprecipitant and
biomolecule
dropwise addition
with stirring
water-miscible
solvent
biomolecule-
coated
microcrystals
The ratio of active mAb to coprecipitant/PSA was varied between 17%w/w and
33 %w/w, as shown in this table (Theoretical Protein Loading (%w/w)).
Theoretical
Protein Loading
(%w/w)
Glycine
(%w/w)
Precipitation
Stabilizing
Additive
(%w/w)
Trehalose
Dihydrate
(%w/w)
mAb Stock (Stored
@ 4-8ºC)
PFCP1_82_6 - No
AdditiveIPA 41.1 57.0 0.0 0.0
PFCP1_82_1 -
Trehalose OnlyIPA 32.0 44.4 0.0 22.0
PFCP1_56_1 IPA 16.8 55.7 4.5 22.3
PFCP1_56_2 IPA 17.2 56.9 13.7 11.4
PFCP1_56_3 IPA 32.7 46.4 19.4 0.0
PFCP1_56_4 2M1P 32.7 46.4 19.4 0.0
PFCP1_56_5 IPA 26.6 37.7 15.7 18.8
PFCP1_56_6 2M1P 26.6 37.7 15.7 18.8
Coprecipitation
Solvent
PCMC Composition (%)
Sample
Stability Under Accelerated Stress Conditions
Monomer Content (%) Storage @ 40 oC for 47 weeks
(Uncontrolled Humidity)
90
91
92
93
94
95
96
97
98
99
100
0.0 10.0 20.0 30.0 40.0 50.0
Time (Weeks)
Monomer Content (%
)
PFCP1_56_2 PFCP1_56_3 PFCP1_56_4 PFCP1_56_5 PFCP1_56_6
Monomer Content (%) Storage @ 25oC for 13 weeks
(Uncontrolled Humidity)
97.5
98
98.5
99
99.5
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (Weeks)
Monomer Content (%
)
PFCP1_56_2 PFCP1_56_3 PFCP1_56_4 PFCP1_56_5 PFCP1_56_6
PFCP PCMC powders were stored at 25ºC in sealed vials at ambient humidity for a standard period of 13 weeks.
All samples retained >98.5% monomer content.
(Liquid PFCP stored @ 2-8ºC had a monomer content of 99.6% monomer.)
PFCP PCMC powders were stored in sealed vials at 40ºC ambient humidity for a standard period of ~47 weeks.
All samples retained ≥95% monomer content. Sample PFCP1_56_6 retained 98.5% monomer - a change of only 1.1% over 47 weeks.
(Liquid PFCP stored @ 2-8ºC had a monomer content of
99.6% monomer.)
Dry PCMC mAb powders incorporating Glu and Arg exhibit high stability under
accelerated stress conditions. Inclusion of a further neutral additive such as
trehalose enhances stability even further.
Monomer Content after Coprecipitation
After drying, the PFCP PCMC material was reconstituted into histidine buffer at a
target protein concentration of 1 mg/mL, and monomer content was measured by
size-exclusion chromatography, using a Tosoh TSKGel G3000 SWXL7.8 mm ID x
30 cm column.
These results show that the mAb remains almost exclusively as monomer when
PCMC coprecipitation is undertaken with Glu and Arg present. When no additives
or trehalose alone were used, significant formation of higher molecular weight
species occurred.
Monomer Content after Solvent Coprecipitation & Reconstitution (%)
99
.7
82.7
90
.87
98
.87
99.3
1
99
.49
99
.5
99.5
2
99
.64
80.081.082.083.084.085.086.087.088.089.090.091.092.093.094.095.096.097.098.099.0
100.0
mA
b S
tock
(Sto
red @
4-
8ºC
)
PF
CP
1_
82_
6 -
No A
dditiv
e
PF
CP
1_
82_
1 -
Tre
halo
se O
nly
PF
CP
1_56_
1 -
PS
A / T
rehalo
se
/ IP
A
PF
CP
1_56_
2 -
PS
A / T
rehalo
se
/ IP
A
PF
CP
1_
56_
3 -
PS
A / IP
A
PF
CP
1_
56_
4 -
PS
A / 2
M1P
PF
CP
1_56_
5 -
PS
A / T
rehalo
se
/ IP
A
PF
CP
1_56_
6 -
PS
A / T
rehalo
se
/ 2M
1P
Monomer Content (%
)
Monomer Content (%) Storage @ 25ºC for 13 weeks
(Uncontrolled Humidity)
Monomer Content (%) Storage @ 40ºC for 47 weeks
(Uncontrolled Humidity)
Monomer Content (%) Storage @ 25ºC for 13 weeks
(Uncontrolled Humidity)
Monomer Content (%) Storage @ 40ºC for 47 weeks
(Uncontrolled Humidity)
