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Special aspects in the pharmaceutical development of protein formats beyond mAbs
MIBio2012 –Molecular Interactions in Biopharmaceutical FormulationsDr. Susanne Jörg, Novartis Pharma AG, BaselBPRD Non-Platform Molecules and Technology, Pharmaceutical Develoopment
New molecule formats and their challenges
Main stability challenges
Increased aggregation
Decreased solubility, precipitation
Protein degradation
Viscosity challenges
Material incompatibility / adsorption
...
Antibody fragments1
Nanobodies2
Therapeutic proteins3
Fusion proteins4
2 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
1 P. Chames. British J Pharmacol. 2009, 157: 220-332 N. Nicolaides. Expert Opin. Drug Discov. 2010, 5(11):1123-403 www.pdb.org4 Novartis, IBP
2
Agenda
Molecule StabilisationCase study on stabilisation of single chain antibody fragment
• pH and buffer screen• Lyophilisate screening study• Surfactant optimisation study
Administration challengesCase studies on administration of biopharmaceutcial formulations
• Drug-drug combinations• Challenges due to material
incompatibilities
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Molecule characteristics
Single-chain variable human-derived monoclonal antibody fragment (scFv)
Prone to covalent and non-covalent aggregation
High molecular weight aggregates, resulting in a not adequately controlled aggregate size distribution (batch to batch variability).
Limited solubility at physiological pH range Source: Stefan Ewert & Barbara Brannetti
Parameter Structural units (examples) Methods
Overall aggregation(non-covalent and covalent)
• SEC, native conditions • DLS, turbidity, subvisible and
visible particlulate matter
Covalent aggregation
• SEC, denaturing conditions• SDS-PAGE, red./non-red.
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Objectives
Development of a stable formulation as lyophilisatein vial for iv infusion
reduced formation of non-covalent aggregation and sub-visible and visible particulates
batch-to-batch consistency
local tolerability of the formulation
• Target pH range based on solubility
• Selection of buffers suitable for target pH
• Target pH range based on solubility
• Selection of buffers suitable for target pH
pH and buffer screen (liquid)
• Evaluation of stabilizers • Evaluation of surfactants• Evaluation of antioxidants
• Evaluation of stabilizers • Evaluation of surfactants• Evaluation of antioxidants
Lyophilisate screening
study
• Establish pH range• Optimise surfactant
concentration
• Establish pH range• Optimise surfactant
concentration
Optimisation study
5 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
pH and Buffer Selection Study Results - non covalent aggregates, particulate analytics
Buffer type• TRIS, glycine and histidine
with comparable non-covalent aggregation behaviour
• Arginine with highest turbidity, subvisble and visible particulate matter
pH value• pH 8.5: increased turbidity,
subvisble and visible particulate matter
• pH of 10.0 brings no significant improvement over pH 9.0 in non-covalent aggregation behaviour
0.0
50.0
100.0
150.0
200.0
250.0
300.0
350.0
8.5 9.0 10.0 8.5 9.0 10.0 8.5 9.0 10.0 8.5 9.0 10.0
50 mM TRIS 50 mM Arginine 50 mM Histidine 50 mM Glycine
Turb
idity
[NTU
]
t=0f/t
t=1w @ 5°C
t=1 w @ RT
6 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
8.5 9.0 10.0 8.5 9.0 10.0 8.5 9.0 10.0 8.5 9.0 10.0
50 mM TRIS 50 mM Arginine 50 mM Histidine 50 mM Glycine
Part
icle
s >1µ
m [p
er m
L]
t=0f/tt=1w @ 5°Ct=1w @ RT
4
pH and Buffer Selection Study Results –degradation products
SDS-PAGE nSEC
Histidine with degradation products• SDS PAGE: additional bands under
reduced and non-reduced conditions• nSEC: Histidine with 2nd peak,
corresponding to a MW of ~5000 Da
Lane 1: MW MarkerLane 2, 7, 12: BlankLane 3-6: non-reduced SDS-PAGE (3: Tris; 4: Arginine; 5: Histidine; 6: Glycine; all pH 9.0 and after 1wk storage @ RT)Lane 8-11: reduced SDS-PAGE (8: Tris; 9: Arginine; 10: Histidine; 11: Glycine; all pH 9.0 and after 1wk storage @ RT)
1 2 3 4 5 6 7 8 9 10 11 12 TRIS, pH 9.0
Histidine, pH 9.0
7
pH and Buffer Selection Study Results – Determination of B22-values by SIC
pH value• B22 values are quite negative below pH 6• Colloidal stability is maximal when the pH is at 8.5 or above. Above that pH,
the response surface for B22 values is relatively flat.Buffer type
• Tris and glycine as best buffers, providing comparable increase in B22 values• As Tris seems to provide better colloidal stability for a wider pH range, it is
recommended to use Tris
TRIS buffer Gly buffer
8 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
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pH and Buffer Selection Study Buffer titration curves
Tris (pKa 8.3) and arginine (pKa 9.0) offer a better buffering capacity towards pH values below pH 9.0 than glycine (pKa 9.6) and histidine (pKa 9.2).
Histidine with high buffering capacity which is disadvantageous at pH value out of physiological range.
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
2 3 4 5 6 7 8 9 10 11 12 13
pH
Volu
me
of a
dded
1N
-NaO
H [m
L]
50mM Arginine
50mM Glycine
50mM TRIS
50mM Histidine
target pH
9 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
Lyophilisate Screening StudyResults - turbidity
StabilizerTrehalose superior over other stabilizers tested.
10
6
Lyophilisate Screening StudyResults - non covalent aggregates, particulate analytics
SurfactantPresence of surfactant prevents increase in turbidity, subvisible particulate matter.Polysorbate 80 superior over other surfactants tested
Antioxidantno advantages.
11 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
Lyophilisate Screening Study Results – Determination of B22-values by SIC
Surfactant• Polysorbate 80 identified as most suitable surfactant to increase B22 values,
while Polysorbate 20 was found to be destabilizing.• Other surfactants tested did not provide any improvement in B22 values. • In addition, combinations of surfactants also do not appear to be promising for
increasing B22 values (not shown)
5.08
-8.61
-5.82
-12
-10
-8
-6
-4
-2
0
2
4
6
8
Tween 80 Tween 20 SDS
Surfactant
B22
x 1
04 (m
ol m
l g-2
)
PS 80 vs. PS20 vs. SDS
12 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
PS 80 vs. NLS vs. Cholates
7
Lyophilisate Screening Study Results – covalent aggregates
Reconstituted Lyo T=0 Monomer [%]
Dimer [%]
Agg.[%]
TRIS Drug substance 88.6 9.7 1.7Polysorbate 80 90.2 9.2 0.0Polysorbate 20 88.9 11.1 0.0Na-desoxycholate 86.8 13.3 0.0PS80 / methionine 88.0 12.1 0.0PS80 / cysteine 67.9 20.3 9.8
dSEC
Cysteine: increase in covalent dimers
Constant pattern for all other surfactants
Peptide Map
Cysteine: formation of non-expected intermolecular disulfide bridges
Ppt2
1-24
-Ppt
89-9
9 (C
23-C
97)
Ppt1
68-1
78, P
pt16
8-17
81 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ]
1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ]
1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ] 12-65 min
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ]
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ]
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ] 65-104 min
10775.01.SR (PS80)10775.09.SR (PS20)10775.15.SR (PS80, Cys)
Ppt8
9-99
-Ppt
155-
160
(C97
-C15
9)Pp
t45-
51Pp
t89-
99-P
pt89
-99
(C97
-C97
) Dim
er
Ppt1
55-1
60-P
pt19
8-23
9 (C
159-
C22
4)
Ppt1
98-2
39-P
pt19
8-23
9(C
224-
C22
4) D
imer
Ppt2
1-24
-Ppt
155-
160
(C23
-C15
9)
Ppt4
0-44
(Q40
, -18
)
Ppt2
40-2
43Pp
t40-
44 (Q
40, -
17)
Ppt2
45-2
55 (M
od.,
+14)
Ppt1
4-20
Ppt2
40-2
44Pp
t245
-255
Ppt2
44-2
55
Ppt1
-13
Ppt1
-13
(N-te
rmin
us a
cety
late
d)
Ppt7
6-85
Ppt5
2-75
Ppt1
82-1
97
Ppt7
6-88
Ppt5
2-88
Ppt8
9-99
-Ppt
198-
239
(C97
-C22
4)Pp
t89-
99-P
pt19
8-23
9 (C
97-C
224,
-18)
Ppt2
5-39
Ppt1
03-1
54Pp
t103
-154
, (M
140,
-18)
Ppt1
82-1
97, (
Mod
,+43
)
***
Ppt1
55-1
60-P
pt15
5-16
0 (C
159-
C15
9 D
imer
)
Ppt2
1-24
-Ppt
89-9
9 (C
23-C
97)
Ppt1
68-1
78, P
pt16
8-17
81 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ]
1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ]
1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 T i m e [m i n ]
0
1 0 0
2 0 0
3 0 0
4 0 0
In te n s.[m A U ] 12-65 min
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ]
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ]
6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 T i m e [ m i n ]0
2 0 0
4 0 0
6 0 0
I n t e n s.[ m A U ] 65-104 min
10775.01.SR (PS80)10775.09.SR (PS20)10775.15.SR (PS80, Cys)
Ppt8
9-99
-Ppt
155-
160
(C97
-C15
9)Pp
t45-
51Pp
t89-
99-P
pt89
-99
(C97
-C97
) Dim
er
Ppt1
55-1
60-P
pt19
8-23
9 (C
159-
C22
4)
Ppt1
98-2
39-P
pt19
8-23
9(C
224-
C22
4) D
imer
Ppt2
1-24
-Ppt
155-
160
(C23
-C15
9)
Ppt4
0-44
(Q40
, -18
)
Ppt2
40-2
43Pp
t40-
44 (Q
40, -
17)
Ppt2
45-2
55 (M
od.,
+14)
Ppt1
4-20
Ppt2
40-2
44Pp
t245
-255
Ppt2
44-2
55
Ppt1
-13
Ppt1
-13
(N-te
rmin
us a
cety
late
d)
Ppt7
6-85
Ppt5
2-75
Ppt1
82-1
97
Ppt7
6-88
Ppt5
2-88
Ppt8
9-99
-Ppt
198-
239
(C97
-C22
4)Pp
t89-
99-P
pt19
8-23
9 (C
97-C
224,
-18)
Ppt2
5-39
Ppt1
03-1
54Pp
t103
-154
, (M
140,
-18)
Ppt1
82-1
97, (
Mod
,+43
)
***
Ppt1
55-1
60-P
pt15
5-16
0 (C
159-
C15
9 D
imer
)
13
Optimisation Study Results: turbidity
Turbidity stays constant around 6-7 NTU for formulations with 0.005 – 0.02% polysorbate 80. Increased turbidity in formulations with 0.001 and no surfactant, particularly upon shaking and freeze-thaw..Turbidty constant for pH 9 – 9.7. Higher turbidity observed for pH 8.7.
14 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
8
Optimisation Study Results – subvisible particulate matter
Particulate matter increase upon lyophilisation. 0.01 – 0.02% PS80 sufficient to keep number of particles ≥ 1.0µm/ml below 10,000.
Particle sizes ≥ 10 and 25 µm within Pharmacopoeial limitsFormulation with 0.005, 0.001 and no surfactant show increase in subvisible particles upon shaking, freeze-thaw and lyophilisation.
15
Optimisation Study Results – DLS
0
5
10
15
20
0.1 1 10 100 1000 10000
Inte
nsity
(%)
Size (r.nm)
Size Distribution by Intensity
0
5
10
15
0.1 1 10 100 1000 10000
Inte
nsity
(%)
Size (r.nm)
Size Distribution by Intensity Lyo T=0Peak 1 [nm]
Peak 2 [nm]
PDI1
Drug Substance 19.7 > 1000 0.22
pH 9.25, 0.02% PS80 16.1 n.a. 0.15
pH 9.25, 0.01% PS80 16.7 > 1000 0.18
pH 9.25, 0.005% PS80 16.5 > 1000 0.16
pH 9.25, 0.001% PS80 17.0 > 1000 0.18
pH 9.25, no PS80 17.3 > 1000 0.20
pH 9.25, 0.02% PS80
pH 9.25, no PS8016 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
1 Polydispersity Index
9
Summary
Selected formulation shows desirable quality upon lyophilization and short-term accelerated stability - reduced non-covalent aggregation as well as sub-visible and visible particulate matter formation.
• pH 9.0 – 9.5 identified as optimal pH range. At lower pH increased molecular weight, turbidity, subvisble and visible particulate matter load.
• Tris and Trehalose superior over other buffers / stabilizers tested.
• Presence of surfactant prevents aggregate formation. Polysorbate 80 significantly superior over other surfactants tested.
| MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs17
• Selection and optimisation of well-known formulation conditions allow keep ing challenging molecule properties under control.
• Variety of analytical methods applied to thoroughly investigate critical quality attributes, such as covalent and non-covalent aggregation and degradation.
Agenda
Molecule StabilisationCase study on stabilisation of single chain antibody fragment
• pH and buffer screen• Lyophilisate screening study• Surfactant optimisation study
Administration challenges• Case studies on administration of
biopharmaceutcial formulations• Drug-drug combinations• Challenges due to material
incompatibilities
18 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
10
Administration challengesDrug-drug combination products
Goals
• Establish drug-drug combination products with enhanced efficacy• Ease of use as a combined single administration
Challenges
• Incompatibilities during storage and use• Analytical challenges in mixtures - distinguishing between 2 similar
molecules and degradants• Each combination project equals effort of 3 single API projects-
individual molecules and combined
Status
• No single unit combo-biologic on the market to date• Several in clinical evaluation
19 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
Drug-drug combination products Delivery options
Mix at point of use• Prerequisite: in-use stability
and combo compatibilityBA + A+B
BA + A B+Sequential administration• easiest option for PoC • potentially long infusion
times
A+BA+B
A+B
Final DP is mixture• Best commercial option• Prerequisite: fixed dose
ratio to be known
A+B
or
Courtesy of T. Serno
20 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
11
Drug-drug combination products Analytical challenges
| MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs21
mAb mix in HP-SEC-chromatogram Overlay of CEX chromatograms
14.91 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.13-1.8
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
65.0
70.0
75.0
min67.2 68.0 68.5 69.0 69.5 70.0 70.5 71.0 71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.2
-16
50
100
150
200
250
300
350
400
450
500
min
21
Main peakmAb A Main peak
mAb B
Aggregation productsof mAb A and mAb B
Insufficient peak resolution for quantification(separation by size), still useful for purityassessement
CEX provides improved peak resolution (separation by charge), suitable for quantification
Main peakmAb B (digested)
Main peakmAb A (digested)
Courtesy of T. Serno
Compatibility issue of mAb derived proteinDescription and interim solution
Compatibility issue Compatibility testing of mAb derived protein with i.v. administration sets• At very low doses adsorption is
problematic (observed at 0.3 mg/kg); • Sub-visible particulate levels formed at
a dose ≤ 1.0 mg/kg during infusion through i.v. line.
Sub-visible particles being identified as proteinaceous particulates.
Interim solutionSpecific release of two i.v. sets which were found compatible.
No pattern observed with respect to line or filter materialse.g. Supplier 1 passed, Supplier 2 failed for PVC/DEHP infusion line
Use of smaller infusion bags to achieve conc. of 0.8-18mg/ml
Courtesy of T. Serno
MFI screenshots of subvisibleparticles
22 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
Protein dose [mg] Infusion volume [ml]
0 – 900 50
80 – 1800 100
200 - 4500 250
12
Compatibility issue of mAb derived proteinRoot cause investigation
Spiking of Surfactant (at a conc.above the CMC of the surfactant) significantly reduced particle formationSpiking of Buffer / Stabilizer without effectDilution of Surfactant was likely the reason for problems encountered during compatibility testing
23 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
0
10
20
30
40
50
Parti
cles
/mL
by L
O
Cumulative particles ≥ 10um
Courtesy of T. Serno
Before spiking ofFilter Surfactant Buffer Stabilizer
0
500
1000
1500
2000
2500
3000
3500
Part
icle
s/m
L by
LO
Cumulative particles > 2µm
Before spiking ofFilter Surfactant Buffer Stabilizer
Administration challengesMitigation options – alternative i.v. infusion modes
Mitigation options Description
1) Partial infusion • Higher protein concentration, but smaller infusion volume• Bag is emptied until defined infusion volume is remaining• Limited by minimal infusion time clinically required for infusion
2) Syringe pump infusion
• Higher protein concentration and low infusion rate• Limited by minimal. clinically acceptable infusion rate
3) Bolus injection • Use of higher protein concentration and small injection volume• Limited by minimal infusion time clinically required for infusion
4) Piggyback infusion • High concentration drug mixed with diluent using «three-waystopcock» just prior infusion into patient
• Requires infusion pump for diluent and syring pump for drug5) Spiking of excipient • Excipient spiked into infusion bag prior to dilution of drug
24 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
13
Conclusions
New biologics molecules– such as mAb derived formats, nanobodies, therapeutic and fusion proteins – come along with new challenges.
• Case Study 1:• Selection of optimised formulation conditions ensures to keep challenging molecule
properties under control. • Variety of analytical methods has been applied to thoroughly investigate critical
quality attributes, such as covalent and non-covalent aggregation and degradation.
Case Study 2: • Drug-drug combination product development need to trade-off between complexity
during clinical development and ease of use for commercial applications.• Compatibility of the protein with materials used during administration to the patient
needs to be assured. Creative solutions might be required to ensure clinical dosing.
Thorough pharmaceutical development is key to enable integrity and stability of biologics compounds upon storage and administration.
25 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs
Acknowledgements
Case Study 1:
Jürgen Sigg, Roland Düblin, Stefan Rapp
Hui Zhao, Kurt Forrer, Manuel Diez
Legacy Biodesign
Case Study 2
Tim Serno, Marco Lang
... and all BPRD Pharmaceutical Development Colleagues
26 | MIBio 2012 | S.Jörg | 30.10.2012 | Special aspects in the pharmaceutical development of protein formats beyond mAbs