Special aspects in the pharmaceutical development of protein formats … · 2017-12-21 · Special aspects in the pharmaceutical development of protein formats beyond mAbs MIBio2012

1

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


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.


3

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


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


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


5

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


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.


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


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


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


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


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.


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


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


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


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)


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


MFI screenshots of subvisibleparticles


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


0

10

20

30

40

50

Parti

cles

/mL

by L

O

Cumulative particles ≥ 10um


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


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.


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


Documents

Special aspects in the pharmaceutical development of protein formats … · 2017-12-21 · Special aspects in the pharmaceutical development of protein formats beyond mAbs MIBio2012