hydroxylpropyl ß-cyclodextrin: a promising excipient … · protein aggregates reduce drug...
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HYDROXYLPROPYL ß-CYCLODEXTRIN: A PROMISING EXCIPIENT FOR PROTEIN
STABILIZATION
DDF 2019, Berlin
Rajeev Gokhale
212 Mar 2019 Global Drug Delivery and Formulation Summit 2019
ABSTRACT
• Protein stability is a major challenge to overcome during manufacturing, upstream and downstream processes, formulation, transportation and storage of biopharmaceuticals. Proteins may aggregate during these activities in addition to chemical changes. Aggregation is often irreversible leading to inactive and potentially immunogenic species, resulting in to reduced efficacy and toxicity to patients. Formulation development involves selection of appropriate excipients to stabilize the protein drugs throughout its recommended shelf life against potential excursions in its life cycle and aid in the delivery of therapeutics into the patient. While role of many excipients in the stability of formulation is well documented, limited information is available on the role of Cyclodextrins in stabilization of therapeutic proteins such mAbs. Cyclodextrins play a vital role in reduction of interfacial tension with the environment and hydrophobic interactions among the protein molecules. In the present study stabilizing effects of novel Cyclodextrins, KLEPTOSE® HPB and HP in various therapeutics protein formulations have been investigated. Physical-chemical stability was influenced under different stress conditions such as thermal and agitation for monoclonal antibodies and human growth hormone. We employed a two-prong approach, high throughput screening and conventional chromatographic methods to establish validation. This study provides insight in to both methodologies to understand interplay between protein and surrounding environments. We attempt to provide the role of novel Cyclodextrins to reduce aggregation and develop therapeutically effective and safe protein formulations
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✓ Proteins are inherently unstableProteins are sensitive to temperature changes, shearing, shaking, solvents, ionic strength, purity, protein concentration, pressure, freeze/thaw-drying cycles. They are generally stable in narrow pH range, and are susceptible to adsorption.
✓ Of the various degradation pathways, aggregation is one of the most common and a cause for great concern.
Protein aggregates reduce drug efficacy and can induce immunogenicity (!)
✓ Formulation is a key element of a successful, stable protein drug. By identifying optimalsolution conditions such as pH, ionic strength, and excipients, the propensity for degradationcan be reduced (Goldberg et al., 2011).
CHALLENGES IN PROTEIN FORMULATIONS
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PROTEINS UNDERGO CHANGES DURING PROCESSES, STORAGE AND TRANSPORTATION
SN Stress Factors
Process/storage/transportation conditions
1 Elevated Temp
Production, improper shipment, storage or handling or deviations, specific production processes like spray drying
2 Freeze-thaw
In process storage, accidental freezing during shipment, Lyophilization
3 Mechanical stress
Production process; shipping (pumping, filtering, stirring, shaking)
4 Light Exposure to day light during production processes, shipping, handling; UV detection during DSP unit operations
5 Oxidative stress
Contacts with oxygen (air, dissolved oxygen), excipients-e.g. peroxides in Polysorbates
6 pH change Production processes (different loading and elution conditions in chromatographic purifications), freezing, formulation
7 Interfaces Air water interface, filters, primary packaging, silicone oil
8 Radiations X-ray in air freight transportation
512 Mar 2019 Global Drug Delivery and Formulation Summit 2019
FORMULATION DESIGN
Buffer
Excipient 3
Tune viscosityExcipient 2
Enhance solubility
Excipient 1 Prevent aggregation
Surfactant Reduce interfacial tension
Ensure initial stability
Solubility
Long-term stability
(aggregation)
Viscositye.g. Polysorbates, poloxamer
e.g. hydroxypropyl cyclodextrin
e.g. arginine
e.g. sugars, polyols, hydroxypropyl cyclodextrin
e.g. citrate, histidine, phosphate
Formulation Challenges
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CONCERNS IN THE USE OF POLYSORBATES
Autooxidation of ethylene oxide
Peroxides
Oxidation of protein
Protein aggregation
❑ Polysorbates are the most widely used non-ionic surfactants
• > 70 % the marketed formulations of monoclonal antibody products contain either polysorbate 20 or 80.
❑ Protect protein pharmaceuticals against interface-induced
aggregation and surface adsorption
❑ Polysorbates 20 and 80 are chemically diverse mixtures• contain mainly sorbitan polyoxyethylene esters of fatty acids• prone to degrade by autooxidation and hydrolysis• presence of residual levels of peroxide in bulk
❑ Polysorbates degradation concerns:
lowered ability of the surfactant to protect the formulation against interfacial stressesimpact of the degradation products on the stability of the protein.
Polyoxyethylenemoieties
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HYDROXYPROPYL-ß-CYCLODEXTRINS
❑ CDs and their derivatives are established enabling excipients in small molecule formulation development
➢ Solubility and bioavailability enhancement
➢ Improve drug chemical and physical stability
➢ Taste-masking
Hydroxypropyl-ß-Cyclodextrin
Hydrophobic Cavity
Hydrophilic Exterior
Unique structure allows for the formation of inclusion
complexes, where lipophilic compounds are
non-covalently bound within the cavity
M.W.
MS, DS
Solubility in
H2O
KLEPTOSE® HPB~1390
0.65, 4.3>50% (20 ºC, w/w%)
KLEPTOSE® HP~1493
0.88, 6.7>50% (20 ºC, w/w%)
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HPßCD – PROTEIN STABILIZING MECHANISMS
❑ HPßCD can act as inhibitor of protein aggregation in liquid formulations by:
i. Binding to the exposed hydrophobic resides on the proteins. This binding lead to shielding of hydrophobic interactions and thereby blocking potential protein-protein interaction which cause aggregation.
➢ The cavity diameter of ß-CDs derivatives allows for a fit of Phe, Tyr, His and Trp into the hydrophobic moiety.
ii. Inhibiting protein aggregation induced by exposure to air-water interface
➢ HPßCD could inhibit protein aggregation in a similar manner to non-ionic surfactants, by displacing proteins from the air-water interface.
➢ HPßCD shows surface activity at concentration of 0.1% (w/v).
12 Apr 2017 Confidential
Protein
Reference : Serno, T., R. Geidobler, and G. Winter, Protein stabilization by cyclodextrins in the liquid and dried state. Adv Drug Deliv Rev, 2011. 63(13): p. 1086-106.
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0
100
200
300
400
500
600
700
800
900
0 50 100 150 200 250
Osm
ola
lity
(mO
smo
l/kg
)
Concentration (mM)
Osmolality of KLEPTOSE®HP/HPB
KLEPTOSE®HP
KLEPTOSE®HPB
▪ Usage of KLEPTOSE HP/HPB at concentrations as high as 250 mM will not impart high viscosity to protein solutions
▪ Acceptable viscosity for SC injections is ~1-20 cps
▪ Recommended upper osmolality limit for SC injection: under 600 mOsm/kg
▪ Usage of KLEPTOSE HP/HPB at high concentrations (i.e. above ~100-200 mM) may be acceptable
PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPB
Osmolality and Viscosity
PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPB
0
20
40
60
80
100
0 100 200 300 400 500V
isco
sity
(m
Pas
)
Concentration (mM)
Viscosity of KLEPTOSE®HP/HPB
KLEPTOSE®HP
KLEPTOSE®HPB
M.W.
MS, DS
Solubility in H2O
KLEPTOSE® HPB (Biopharma grade)~1390
0.65, 4.3>50% (20 ºC, w/w%)
KLEPTOSE® HP (Biopharma grade)~1493
0.88, 6.7>50% (20 ºC, w/w%)
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PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPBSurface Tension
50
55
60
65
70
75
0 50 100 150 200 250
Surf
ace
Ten
sio
n (
mN
/m)
Concentration (mM)
Surface tension of KLEPTOSE®HP/HPB
KLEPTOSE®HP
KLEPTOSE®HPB
MS~0.88
▪ KLEPTOSE®HP/HPB showed surface-active properties.▪ Higher HP substitution, lower surface tension. ▪ Critical concentration of KLEPTOSE®HP is ~ 3-4 mM and HPB is ~ 10 mM.▪ Critical micellar concentration (CMC) of polysorbate 80/20 is 0.012 mM and 0.08mM,
respectively
MS~0.65
1111
• High throughput: Multiple stability and conformational parameters from one sample
AGGREGATION STUDY OF KLEPTOSE® HP & HPB: NANODSF
Drawbacks: Study aggregation profiles of protein at thermal unfolding temperatures (i.eabove Tm). Protein aggregation propensity might be different in real-time stability studies.
Fluorescence detection at 350 nm and 330 nm→ measures protein unfolding profile
Light scattering → measures relative amount of aggregation
DSF and Aggregation profiles of mAb in different formulations
Unfolding profiles
Tm’sOnset T of Aggreg.
F 3
50
/33
0 r
atio
Bac
k Sc
atte
rin
g
Relative amount of aggregation
Temp. ramp
1212
• Thermal ramp analysis using nanoDSF
https://www.researchgate.net/figure/Infliximab-Remicade-protein-structure_fig1_303483394
Human Growth Hormone (hGH)22 kDa
Infliximab149 kDa
PREVIOUS STUDY : Evaluation of aggregation reduction by KLEPTOSE® HP & HPB
Ba
ck S
ca
tte
rin
g (
mA
U)
Ba
ck S
ca
tte
rin
g (
mA
U)
➢ Suppression of aggregation observed in the presence of KLEPTOSE® HP & HPB
https://www.researchgate.net/figure/Infliximab-Remicade-protein-structure_fig1_303483394Pavlovsky et al. The crystal-structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept Lett. 1995;2:333–340.
Increasin
g HP
B
hGH Agg at 85°CInfliximab Agg at 90°C
Temperature Temperature
Buffer HP 100 mM HPB 100 mM Buffer HP 100 mM HPB 100 mM
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https://www.drugbank.ca/drugs/DB00112
CASE STUDY 1: PROTEIN STABILIZATION BY KLEPTOSE ® HPB & HP
Objective: To benchmark KLEPTOSE ® HP/ HPB against polysorbates in commercial monoclonal
antibodies formulations.
Human Plasma IgG~150 KDa
Model proteins:
Commercial formulation:25 mg/mL bevazizumab, 60 mg/mL α,α-
trehalose dihydrate, 50 mM phosphate buffer, pH 6.2, 0.04 % w/v polysorbate 20
▪ Marketed as Avastin, is a recombinant
humanized monoclonal antibody (IgG1) used to treat a number of types of cancers
Bevacizumab149 KDa
https://www.drugbank.ca/drugs/DB00112
▪ Immunoglobulin G (IgG) is the most abundant
immunoglobulin in plasma
Proposed base buffer used in study:25 mM phosphate buffer, pH 6.8
https://www.differencebetween.com/difference-between-igm-and-vs-igg/
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EXPERIMENTAL PLAN
Controls
Buffer only
0.05% Tween 80
with [HPßCD]
Shaking @ 1400 rpm Incubation at 40 °C
1) NanoDSF:▪ Aggregation Onset
Temperature ▪ Relative amount of
aggregation
2) HPLC-SEC ▪ % soluble monomers,
aggregates & fragments
Human plasma IgG:
Bevacizumab (Avastin):Controls
CF
CF w/o Tween 20 + [HPßCD]
5 mM
20 mM
100 mM
Formulation Strategy: ➢ All formulations will be formulated at 5 mg/mL protein
concentration ➢ For Human plasma IgG, polysorbate 80 (Tween 80) at 0.05
%w/v level (above CMC) will be used as control➢ For bevacizumab, commercial formulation (CF) with/ without
Tween 20 will be used as controls. ➢ Effect of KLEPTOSE ® HP/ HPB was studied at 3 different levels
(i.e 5 mM, 20 mM and 100 mM)
CF
w/o Tween
20
5 mM
20 mM
100 mM
Two-pronged approach
1512 Mar 2019 Global Delivery and Formulation Summit 2019
ANALYTICAL METHODS
Fluorescence detection at 350 nm and 330 nm→ measures unfolding profile
Temperature Ramp: 20 to 95°C at 1.5°C per min
❑ NanoDSF Simultaneous detection
Light scattering → measures relative amount of aggregation
❑ SEC-HPLC
Equipment: Waters Acquity Arc HPLC/PDA detector/ Empower 3
Column: Xbridge® BEH200Å SEC 3.5μm 7.8 x 300mm
Mobile phase: 25 mM sodium phosphate, 150 mM NaCl, pH 6.8
Running condition:
Isocratic, Wavelength 218 nm 0.5 mL/min for 30 min
Injection volume: 10 µL
Prometheus NT.48
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▪ Significant ↓ in aggregation at high KLEPTOSE®HP& HPB concentration
50
100
150
200
250
300
350
400
450
20 40 60 80 100Bac
k Sc
atte
rin
g (m
AU
)
Temperature (°C)63.0°C
64.0°C
65.0°C
66.0°C
67.0°C
68.0°C
69.0°C
Buffer only 0.05 %Tween 80
5 mMHPBCD
20 mMHPBCD
100 mMHPBCD
T_A
gg (
°C)
Aggregation Onset Temperature (°C)KLEPTOSE HP
KELPTOSE HPB
Buffer, Tween 80, 5mM, 20 mM
100mM
Aggregation profiles of protein in the presence of KLEPTOSE® HP & HPB
NanoDSF RESULTS
50
100
150
200
250
300
350
400
450
20.0 40.0 60.0 80.0
Bac
k Sc
atte
rin
g (m
AU
)
Temperature (°C)
Controls, 5mM, 20 mM
100 mM
❑ Bevacizumab
❑ Human plasma IgG
Aggregation profiles
65.0
66.0
67.0
68.0
69.0
70.0
71.0
CF CF w/oTween 20
5mMHPBCD
20mMHPBCD
100mMHPBCD
T_A
gg.(
°C
)
Aggregation Onset Temperature (°C)
KLEPTOSE HP
KLEPTOSE HPB
1712 Mar 2019 Global Delivery and Formulation Summit 2019
NANODSF RESULTS
Thermal unfolding of IgG
0.740
0.745
0.750
0.755
0.760
0.765
0.770
Bufferonly
0.05 %Tween
80
5 mMHP
20 mMHP
100 mMHP
5 mMHPB
20 mMHPB
100 mMHPB
F35
0/3
30
@ 2
5°C
T0
96 hShaking
0.75
0.77
0.80
0.82
0.85
0.87
0.90
0.92
0.95
20 30 40 50 60 70 80 90
F35
0/3
30
Temperature (°C)
% unfolded
IgG Thermal unfolding curve
• Lower % unfolding observed in formulations containing KLEPTOSE HP/HPB
• KLEPTOSE HP/HPB prevent unfolding of IgG from agitation stress
❑ Effect of agitation stress
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HUMAN PLASMA IgGAgitation Stress
❑Effect of KLEPTOSE HP/HPB on agitation-induced aggregation
• % soluble monomer was higher in formulations with KLEPTOSE® HP/HPB compared to control or Tween 80
• High concentration of KLEPTOSE® HP/HPB (i.e. 100 mM) was able reduce the rate of aggregation
11.5
12
12.5
13
Frag
men
t (%
)
% FragmentsT0
Shake 48 h
Shake 96 h
1.5
2.0
2.5
3.0
3.5
4.0
Solu
ble
Agg
rega
te (
%)
% Soluble aggregates
T0
Shake 48 h
Shake 96 h
• Longer duration of agitation resulted in fragmentation of monomer, which could be inhibited by KLEPTOSE® HP/HPB
Buffer
only
0.0
5%
Tw
een-8
0
5 m
M H
P
20 m
M H
P
100 m
M H
P
5 m
M H
PB
20 m
M H
PB
100 m
M H
PB
8 08 59 09 5
9 5
9 6
9 7
9 8
9 9
1 0 0
% S o lu b le m o n o m e r
T0
S h a k e 4 8 h
S h a k e 9 6 h
So
lub
le m
on
om
er
(%)
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BEVACIZUMABAgitation Stress
❑ Effect of KLEPTOSE® HP/HPB on agitation-induced aggregation
➢ Bevacizumab is stable towards agitation. Minimal increase in protein aggregation was observed after extensive shaking.
➢ However, it was observed that the rate of aggregation was lower in formulations containing KLEPTOSE®HP& HPB as compared to the commercial formulation.
➢ KLEPTOSE ® HP& HPB may possibly serve as an alternative to polysorbates in bevacizumab formulations
1.8
2.0
2.2
2.4
2.6
2.8
CF CF w/oTween
20
5 mMHP
20 mMHP
100 mMHP
5 mMHPB
20 mMHPB
100 mMHPB
Solu
ble
Agg
rega
te (
%)
Soluble Aggregate (%)
T0
24 h Shaking
96 h Shaking
2012 Mar 2019 Global Delivery and Formulation Summit 2019
HUMAN PLASMA IgG
Heat Stress
❑Effect of KLEPTOSE HP/HPB on heat-induced aggregation
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
% S
olu
ble
agg
rega
te
Soluble Aggregate (%)
T0
1 week @40 deg C
+1.58 +1.67 +1.21 +1.36 +1.23 +0.82 +1.25 +1.13 +0.75
➢ KLEPTOSE® HP and HPB reduced heat-induced aggregation in a concentration-dependent manner.
➢ KLEPTOSE® HP and HPB when used at optimized concentrations can be as effective as trehalose in inhibiting protein aggregation.
Note: Numbers above chart represent the increase in soluble aggregate after heat stress
2112 Mar 2019 Global Delivery and Formulation Summit 2019
REPRESENTATIVE SEC CHROMATOGRAMS
Human plasma IgG
➢ Human plasma IgG in buffer before and after 1 week heat stress at 40 °C
Aggregate
Monomer ----- T0----- 1 week @ 40 °C
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1.80
2.00
2.20
2.40
2.60
CF CF w/oTween 20
5 mMHPBCD
20 mMHPBCD
100 mMHPBCD
Solu
ble
Agg
rega
te (
%)
soluble aggregate (%)KLEPTOSE HP
KLEPTOSE HPB
1.00
1.50
2.00
2.50
3.00
3.50
Buffer only 0.05%Tween-80
5 mMHPBCD
20 mMHPBCD
100 mMHPBCD
% S
olu
ble
Agg
rega
te
soluble aggregate (%)
KLEPTOSE HP
KLEPTOSE HPB
REVERSIBLE AGGREGATE REDUCTION
➢ Soluble aggregate (%) decreased with addition of KLEPTOSE® HP/HPB➢ Hypothesis: KLEPTOSE® HP & HPB may have the ability to interfere with the reversible self-
association of protein monomers. ➢ Hypothesis requires additional studies to confirm.
❑ Evaluation of reversible aggregate reduction property of KLEPTOSE® HP & HPB
➢ KLEPTOSE HP& HPB were added into unstressed formulations of human plasma IgG and bevacizumab at three different concentrations
➢ % soluble aggregate was measured using SEC .
❑ Human plasma IgG ❑ Bevacizumab
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GENERAL CONCLUSIONS
Case Study
➢ KLEPTOSE® HPB & HP are surface active, may serve as alternative surfactants in protein formulations
➢ Anti-aggregation properties of KLEPTOSE® HP & HPB confirmed using orthogonal methods (nanoDSF & SEC):
➢ IgG
❑Reduce rate of aggregation during agitation and heat stress
❑Prevent fragmentation during agitation
❑Reduce protein unfolding
➢Bevacizumab
❑Reduce rate of aggregation during agitation
❖ KLEPTOSE® HPB & HP are promising excipients that can reduce therapeutic protein
aggregation induced by physical stresses, minimizing protein and efficacy loss
❖ KLEPTOSE® HPB & HP can serve as an alternative option to presently used excipients,
or as a tool to synergistically complement them in biologic formulations
2412 Mar 2019 Global Delivery and Formulation Summit 2019
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