Oxidation of a Glycosylated Therapeutic Protein – Impact on Formulation Stability and Aggregation

Jimmy Smedley, Ph.D. Group Leader, Biopharmaceutical Development

KBI Biopharma, Inc.

Formulation Strategies for Protein Therapeutics

Tuesday, November 1, 2011

Long Beach Convention Center Long Beach, CA

Challenges for Non-Antibody Proteins

• Extraordinarily diverse group of molecules • Enzymes • Interferons • Insulins • Blood Factors • Colony Stimulating Factors • Cytokines • Growth Factors • Conjugates and Fusion Proteins

» Albumins, Enzymes, Antibody fragments • PEGylated and other modified proteins

http://en.wikipedia.org/wiki/Insulin

• Molecular weight • Sizes ranging from a few kDa to >500 kDa

• Secondary, tertiary, and quaternary structure • From relatively unordered to complex, highly ordered,

multimeric states

• Post-translational modifications • Glycosylation state ranging from un-glycosylated to complex,

highly (sometimes variable) glycosylated states

• Activity • Frequent presence of structurally labile, solvent exposed

active sites or effector sites • Diverse range of functions, activity assays ranging from

simple to complex • Activity assay available and suitable for formulation studies?

Challenges of Non-Antibody Proteins

Diverse process streams & process-related impurity profile

• Host cell proteins: E, coli, yeast, mammalian, insect, plant expression systems

• Unique process steps that impact formulation and analytics » PEGylation, conjugation, etc.

• Diverse and often poorly-understood degradation pathways and product-related impurity profile

• Unfolding, aggregation, fragmentation, oxidation, deamidation, disulfide exchange, isomerization

» What is critical to maintain potency?

Challenges of Non-Antibody Proteins

Early Development Challenges • Lack of Platform Analytics and Process • Timing of Analytical Development relative to

Process Development and Formulation Development

• Chicken vs. Egg • Often do not have suite of orthogonal, stability-

indicating analytical and potency methods to support initial formulation & stability studies

• Often resort to default/standard formulations

• Typically given very tight timelines to develop a stable Phase I formulation

“Research Phase”

Biophysical Screen • Identify Critical

Factors • Eliminate Non-

Critical Factors

Solubility DOE &

Accelerated Stability

Forced Degradation

Protein Preformulation Workflow

“Research Phase” – Structural Considerations

Characteristic Information pI 6.3

Molecular Weight ~50 kDa (with glycan)

Glycosylation State N-linked glycan, multiply sites, varying degree, composition, sialic acid content

Secondary/Tertiary Structure

Contains conserved ‘serpin-fold’ with 3 beta sheets surrounded by 8/9 alpha helices.

Mechanism of Action Serine proteinase inhibitor with mouse-trap like conformational change.

http://en.wikipedia.org/wiki/Image:Atatchy.png

• Desired Formulation Type • Liquid, Lyophilized Powder, Other

• Route of Administration • IV, SC, IM, etc.

• Buffer & Formulation pH Selection • Identify suitable buffers based on pKa relative to pI • Solubility considerations • Compatibility with final dosage form

» Buffer type (e.g., lyophilization considerations) • Compatibility with route of administration

» pH and buffer type (e.g., SC injection)

“Research Phase” – Formulation Considerations

Preformulation “Research Phase” • Excipient Selection

• Polyols and Sugars: Solubility, Thermal Stability, Chemical Stability

• Salt: Solubility, Ionic strength, Osmolality • Amino Acids: Solubility, Viscosity • Surfactants: Aggregates and Particulates • Specific ions or factors required for activity or maintenance of

structure

Use of Biophysical Techniques • Evaluate the suitability of “platform-able” biophysical

techniques to support early formulation studies • Generally able to employ standard analysis

parameters to a broad range of proteins • No extensive method development required • Able to gather data quickly on formulation compatibility

• Characterize the thermal and conformational properties of the protein

• Evaluate the impact of formulation factors on thermal, conformational, and physical stability

• Buffer, pH, excipients, surfactants, protein concentration

Initial Biophysical Screening

• To limit the number runs to be evaluated as a part of the DOE study

• Utilize a combination of biophysical tools

• DSC: Thermal/conformational stability • DLS: Aggregation and polydispersity

• Take advantage of orthogonal techniques to make decisions about formulation factors

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Size Distribution by Intensity

Record 100: Sample B Average Record 105: Sample G Average

Buffer Screen – DLS Analysis

Red trace = Glutamate, pH 4.5 Green trace = Phosphate, pH 6.5

• Screened 17 different buffer/pH types

Buffer Screen – DLS Analysis • Possible advantage for lower pH values • Used data to down-select buffer types for excipient screen

Buffer Screen – DSC Analysis • Formulation buffer in reference cell • Formulated protein in sample cell • Buffer scan subtracted from samples scan and integrated • Single transition noted for all samples

20 40 60 80 100 120-0.00057-0.00056-0.00055-0.00054-0.00053-0.00052-0.00051-0.00050-0.00049-0.00048-0.00047-0.00046-0.00045-0.00044-0.00043-0.00042-0.00041 m020509010dsc_cp

m020509011dsc_cp

Cp(c

al/o C)

Temperature (oC)

57.69478

20 30 40 50 60 70 80 90 100 110 120

-20

-15

-10

-5

0

Cp (k

cal/m

ole/

o C)

Temperature (oC)

Buffer scan

Sample scan

Buffer-subtracted scan

Buffer Screen – DSC Analysis • Data sorted by pH • Clear trend with pH and Tm

Excipient Screen – DLS Analysis • Screened 5 different excipients with 3 buffer/pH systems • NaCl and mannitol showed optimal responses for monomer peak

width.

Excipient Screen – DSC Analysis • Data sorted by buffer type 62.38244

20 30 40 50 60 70 80 90 100 110 120-25

-20

-15

-10

-5

0

5

w030909011dsc_cp Peaks1_Pky

Cp (k

cal/m

ole/

o C)

Temperature (oC)

Results from Initial Screen • Buffer screen

• Screened 17 different buffer/pH types • Lower pH’s preferred • Acetate, histidine, glycolate selected

• Excipient screen • Screened 5 different excipients in 3 buffer/pH systems • DLS data showed NaCl, mannitol, sorbitol to be preferred • DSC data demonstrated trehalose and sucrose to have optimal

responses • Surfactant screen

• Freeze-thaw, agitation stress in samples with and without 0.02% polysorbate 80

• Addition of polysorbate was not beneficial to physical or thermal stability

• Note that heat stress had not been performed to this point

Initial Design of Experiments • Buffers

• Acetate (pKa = 4.8) • pH’s 4.5, 5.0, 5.5

• Histidine (pKa = 6.0) • pH’s 5.5, 6.0, 6.5

• Excipients • NaCl, mannitol, trehalose

• Replicates • All pH center-points were in prepared in triplicate • All pH axial points were prepared in duplicate

• BDS exchanged via centrifugal UFDF into each of 36 buffers • Testing

• Time-zero: A280, DSC, DLS, Viscosity*, osmolality* • Four-week (5°C and 45°C): A280, SEC, RP, IEX, DLS*,

Activity* • *only selected samples

pH Buffer type Excipient6.5 20mM Histidine 250mM Mannitol5.5 20mM Acetate 250mM Mannitol6 20mM Histidine 125mM NaCl5 20mM Acetate 250mM Mannitol6 20mM Histidine 250mM Trehalose

5.5 20mM Histidine 125mM NaCl5.5 20mM Histidine 250mM Trehalose5.5 20mM Histidine 250mM Trehalose6.5 20mM Histidine 250mM Mannitol6.5 20mM Histidine 250mM Trehalose5.5 20mM Histidine 250mM Mannitol6.5 20mM Histidine 250mM Trehalose5.5 20mM Acetate 125mM NaCl6.5 20mM Histidine 125mM NaCl5.5 20mM Histidine 125mM NaCl4.5 20mM Acetate 250mM Trehalose5 20mM Acetate 250mM Trehalose6 20mM Histidine 250mM Mannitol

6.5 20mM Histidine 125mM NaCl6 20mM Histidine 250mM Trehalose

4.5 20mM Acetate 125mM NaCl6 20mM Histidine 125mM NaCl5 20mM Acetate 250mM Mannitol

4.5 20mM Acetate 125mM NaCl5 20mM Acetate 125mM NaCl

4.5 20mM Acetate 250mM Trehalose5.5 20mM Acetate 250mM Mannitol4.5 20mM Acetate 250mM Mannitol5.5 20mM Acetate 250mM Trehalose5.5 20mM Acetate 125mM NaCl6 20mM Histidine 250mM Mannitol

5.5 20mM Histidine 250mM Mannitol5.5 20mM Acetate 250mM Trehalose5 20mM Acetate 125mM NaCl5 20mM Acetate 250mM Trehalose

4.5 20mM Acetate 250mM Mannitol

First DOE, t=4W testing • Unstressed samples (5°C) had normal appearance in vials • All stressed samples (45°C) had visible white precipitate at bottom of vials

• UV/Vis analysis

-5.00E-01

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

3.00E+00

3.50E+00

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF GG HH II JJ

A320

5°C 45°C 45°C (sup)

First DOE, t=4W SEC testing

min0 2.5 5 7.5 10 12.5

mA

U0

2040

60

7.390

9.773

MWD1 E, Sig=280,16 Ref=off (CHEMSTOR\6857321\A622843\70109065.D)

min0 2.5 5 7.5 10 12.5

mA

U0

2.5

5.3925.6

00

6.2796.5

29

7.389

9.1329

.6129.9

9210

.186

10.39

6

10.72

1

10.87

9

11.05

9

11.33

411

.990

12.27

1

12.52

1

12.76

9

13.39

9

1

MWD1 E, Sig=280,16 Ref=off (CHEMSTOR\6857370\A623171\70109066.D)

min0 2.5 5 7.5 10 12.5

mA

U0

4080

5.671

6.625

7.420

10.92

4

MWD1 E, Sig=280,16 Ref=off (CHEMSTOR\6851194\A602140\70109007.D)

min0 2.5 5 7.5 10 12.5

mA

U0

50

5.648

6.631

7.419

10.75

9

MWD1 E, Sig=280,16 Ref=off (CHEMSTOR\6851232\A602468\70109008.D)

min0 2.5 5 7.5 10 12.5

mA

U0

1

6.617

7.385

9.096

9.618

10.13

1

10.72

5

11.63

112

.426 14

6

MWD1 E, Sig=280,16 Ref=off (CHEMSTOR\6857747\A623531\70109067.D)

BDS (Ref Std)

Histidine/mannitol, pH 6.5 Acetate/mannitol, pH 5.5

Unstressed

Stressed

Unstressed

Stressed

Study Redesign

• Atypical results prompted further evaluation.

• Heat-stress temperature too severe? • 45°C picked based on DSC scans

• Single cysteine present in protein

• Oxidation-mediated dimerization possible root cause.

• Scouted lower heat-stress temperatures

• Screened buffers with/without addition of

methionine

57.69478

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Cp (k

cal/m

ole/

o C)

Temperature (oC)

Buffer-subtracted scan

Heat Stress Study – SEC Testing

99.1

99.2

99.3

99.4

99.5

99.6

99.7

99.8

99.9

100.0

100.1

2 days 5 days 9 days 14 days

%M

ain

Peak

Are

a

25°C 30°C 37°C

• BDS stored at 25°C, 30°C, and 37°C for 2, 5, 9, 14 days • SEC testing performed to assess HMW formation.

• Only samples exposed to elevated temperatures showed HMW formation

Methionine Evaluation – SEC Testing • BDS buffer exchanged into 4 buffers +/- L-methionine • SEC testing performed to assess HMW formation after limited heat stress

0.0

20.0

40.0

60.0

80.0

100.0

120.0

A B C D E F G H I(BDS)

- 2mM Methionine

- 2mM Methionine

- 2mM Methionine

- 2mM Methionine

20 mM Histidine pH 6.5 20 mM Citrate pH 6.5 20 mM Phosphate pH 6.5 20 mM Carbonate pH 6.5 Current Formulation

Buffer

%M

ain

Peak

Are

a

2W/5C 2W/40C

• Buffers containing methionine had higher %Main Peak Areas. • Methionine appears to be important as antioxidant to prevent aggregation.

Second Preformulation DOE

Formulation pH Buffer Excipient

A 6.5 20mM Histidine 125mM NaCl+2mM MethionineB 7 20mM Histidine 125mM NaCl+2mM MethionineC 6 10mM Citrate 125mM NaCl+2mM MethionineD 6.5 20mM Histidine 250mM Trehalose+2mM MethionineE 6 20mM Histidine 250mM Trehalose+2mM MethionineF 6.5 10mM Citrate 125mM NaCl+2mM MethionineG 6.5 20mM Histidine 250mM Trehalose+2mM MethionineH 6.5 10mM Citrate 250mM Trehalose+2mM MethionineI 7 10mM Citrate 125mM NaCl+2mM MethionineJ 7 10mM Citrate 250mM Trehalose+2mM MethionineK 6 10mM Citrate 250mM Trehalose+2mM MethionineL 6 20mM Histidine 125mM NaCl+2mM MethionineM 6.5 10mM Citrate 125mM NaCl+2mM MethionineN 6.5 20mM Histidine 125mM NaCl+2mM MethionineO 6.5 10mM Citrate 250mM Trehalose+2mM MethionineP 7 20mM Histidine 250mM Trehalose+2mM Methionine

Q 6.5 20mM Histidine 5mM MethionineR 6.5 20mM Histidine 10mM MethionineS 6.5 10mM Citrate 5mM MethionineT 6.5 10mM Citrate 10mM Methionine

Off-DOE Compositions

• Histidine and Citrate as buffers • pH 6.0, 6.5, 7.0 for both

• NaCl and Trehalose as excipients • All formulations contained methionine • More conservative approach used

• Stress temperature = 40°C • More analytics earlier

• t=0 testing • A280 • DLS

• t=2W testing • SEC

• t=4W testing • A280 • DLS • SEC • Activity • IEX

2nd DOE, t=0 – DLS testing • DLS analysis performed on all t=0 samples

• t=0 data show preference for citrate and NaCl.

2nd DOE, t=2W – SEC testing • Early monitoring of physical state at 2W • SEC analysis performed on heat-stressed samples

• As seen at t=0, citrate buffers support lower aggregation than histidine buffers

min0 2.5 5 7.5 10 12.5m

AU

050

100

5.575

7.423

MWD1 A, Sig=280,16 Ref=off (CHEMSTOR\7459832\A923609\10000008.D)

min0 2.5 5 7.5 10 12.5

mA

U0

2040

6080

5.660

7.421

MWD1 A, Sig=280,16 Ref=off (CHEMSTOR\7461322\A926437\10000015.D)

D – His/Tre/Met, pH 6.5 H – Cit/Tre/Met, pH 6.5

2nd DOE, t=2W – SEC testing • All 2W SEC data for %HMW Peak Area

2nd DOE, t=4W – DLS testing • Analyzed both heat-stressed and control samples via DLS, SEC, IEX, and Activity

0

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0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Size Distribution by Intensity

Record 209: 10mM Citrate, 125mM NaCl, 2mM Met, pH 7.0, 2-8CRecord 229: 10mM Citrate, 125mM NaCl, 2mM Met, pH 7.0, 40C

• DLS results show high aggregation after heat stress

2nd DOE, t=4W – DLS testing

0

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0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Size Distribution by Intensity

Record 201: 20mM His, 125mM NaCl, 2mM Met, pH 6.5, 2-8CRecord 221: 20mM His, 125mM NaCl, 2mM Met, pH 6.5, 40C

• Analyzed both heat-stressed and control samples via DLS, SEC, IEX, and Activity

• DLS results show high aggregation after heat stress

2nd DOE, t=4W – DLS testing • Analysis of “Peak 1” diameter shows drastic differences in hydrodynamic radius

2nd DOE, t=4W – SEC testing • SEC testing provided quantitative purity analysis • HMW seen for most heat-stressed samples.

Cit/NaCl/Met, pH 6.5, 5°C

min0 5 10m

AU0

2040

60

5.540

7.376

DAD1 A, Sig=280,16 Ref=off (CHEMSTOR\7541906\A913500\10220938.D)

min0 5 10

mAU

050

7.363

DAD1 A, Sig=280,16 Ref=off (CHEMSTOR\7540565\A892828\10220910.D)

Cit/NaCl/Met, pH 6.5, 45°C

2nd DOE, t=4W – SEC testing • All 5°C samples had 100% Main Peak Area

2nd DOE, t=2W – AEX testing • AEX testing provided quantitative analysis of charge variants • Increase in %Acid Peak Area seen for heat-stressed samples.

His/NaCl/Met, pH 7.0, 5°C

min0 10 20 30 40m

AU

010

11.18

8

35.30

7

MWD1 C, Sig=280,16 Ref=360,100 (CHEMSTOR\7593026\A754109\1CF-3001.D)

min0 10 20 30 40

mA

U0

1020

30

11.10

1

MWD1 C, Sig=280,16 Ref=360,100 (CHEMSTOR\7573175\A738921\M1029095.D)

His/NaCl/Met, pH 7.0, 45°C

2nd DOE, t=4W – AEX testing • All 5°C samples had 100% Main Peak Area

Formulation Optimization • Several rounds of optimization performed • Responses that fit models:

• 2W SEC (%Main Peak Area, %HMW Peak Area) • 4W SEC (%Main Peak Area, %HMW Peak Area) • 4W AEX (%Acidic Peak Area)

• DLS, Activity not included in DOE analysis

ConstraintsName Goal Lower Limit Upper Limit Lower WeigUpper WeigImportancepH is in range 6 7 1 1 3Buffer is in range 20mM Histidine 10mM Citrate 1 1 3

Excipient is in range 125mM NaCl+2mM Methionine 250mM Trehalose+2mM Methionine 1 1 3

(2 Wk 40C SEC Main)^3 maximize 41243.45632 958790.5401 1 1 3Ln(2 Wk 40C SEC HMW) minimize 0.331432415 4.181282077 1 1 3(4 Wk 40C SEC Main%)^2.24 maximize 244.4020035 28451.06886 1 1 3Ln(4 Wk 40C SEC HMW%) minimize 0.96598601 4.481369253 1 1 3(4 Wk 40C AEX Main%)^1.51 maximize 91.15590164 1017.335766 1 1 3Ln(4 Wk 40C AEX Deg%) minimize 0.638374683 4.38383702 1 1 3

Solutions for 4 combinations of categoric factor levelsNumber pH Buffer Excipient (2 Wk 40C S Ln(2 Wk 40C (4 Wk 40C SE Ln(4 Wk 40C (4 Wk 40C A Ln(4 Wk 40C Desirability

1 7 10mM Citrate 250mM Trehalose+2mM Methionine 961235.56 0.4049954 28386.567 1.0641099 1005.7554 1.1404562 0.96618452 7 10mM Citrate 125mM NaCl+2mM Methionine 853670.61 1.5583543 24022.302 2.2049926 922.9455 1.7498581 0.7697263 6 20mM Histidine 125mM NaCl+2mM Methionine 606040.78 2.7052374 24842.931 2.3017205 997.72169 0.8353071 0.70068544 6 20mM Histidine 250mM Trehalose+2mM Methionine 191004.01 3.7557451 7748.2155 3.825674 96.880204 3.2253174 0.1093682

Formulation Optimization • 2nd round of optimization • SEC data at 4W increased in Importance

Name Goal Lower Limit Upper Limit Lower Weight Upper We ImportancepH is in range 6 7 1 1 3Buffer is in range 20mM Histidine 10mM Citrate 1 1 3

Excipient is in range

125mM NaCl+2mM Methionine

250mM Trehalose+ 2mM Methionine

1 1 3(2 Wk 40C SEC Main)^3 maximize 41243.45632 958790.5401 1 1 5Ln(2 Wk 40C SEC HMW) minimize 0.331432415 4.181282077 1 1 5(4 Wk 40C SEC Main%)^2.24 maximize 244.4020035 28451.06886 1 1 5Ln(4 Wk 40C SEC HMW%) minimize 0.96598601 4.481369253 1 1 5(4 Wk 40C AEX Main%)^1.51 maximize 91.15590164 1017.335766 1 1 3Ln(4 Wk 40C AEX Deg%) minimize 0.638374683 4.38383702 1 1 3

Solutions for 4 combinations of categoric factor levelsNumber pH Buffer Excipient (2 Wk 40C SEC Ln(2 Wk 40 (4 Wk 40C SEC Ln(4 Wk 40 (4 Wk 40C Ln(4 Wk 40 Desirability

1 7 10mM Citrate 250mM Trehalose+2mM Methionine 961235.5644 0.404995 28386.5671 1.06411 1005.755 1.140456 0.9727252792 7 10mM Citrate 125mM NaCl+2mM Methionine 853670.605 1.558354 24022.3018 2.204993 922.9455 1.749858 0.7659515683 6 20mM Histidine 125mM NaCl+2mM Methionine 606040.7771 2.705237 24842.9311 2.301721 997.7217 0.835307 0.6672319154 6 20mM Histidine 250mM Trehalose+2mM Methionine 191004.012 3.755745 7748.21548 3.825674 96.8802 3.225317 0.125935884

• Optimizations resulted in final formulation recommendation of 10 mM citrate, 250 mM trehalose, 2 mM methionine, pH 7.0

Experimental Conclusions and Lessons Learned • Heat stress not included as part of baseline

biophysical screen.

• Samples in DOE on accelerated stability exhibited severe aggregation of protein in most formulations.

• Follow up excipient screening demonstrated addition of Met to be beneficial.

• Second DOE set up with Met as second excipient in all formulations.

• Careful monitoring of physical stability of protein during accelerated stability helped prevent catastrophic loss of data set.

• Optimizations resulted in final formulation recommendation of 10 mM citrate, 250 mM trehalose, 2 mM methionine, pH 7.0

http://en.wikipedia.org/wiki/Image:Atatchy.png

Acknowledgements

• Tim Kelly, Ph.D. • VP, Biopharmaceutical Development

• Pooja Arora, Ph.D. • Former Group Leader, Biopharmaceutical

Development • Atul Saluja, Ph.D.

• Former Group Leader, Biopharmaceutical Development