Platform Techniques for Preformulation Development for Non-Antibody Products

Platform Techniques for Preformulation Development for

Non-Antibody Products2009 IBC Non-Antibody Protein Therapeutics

Development and Production

Tim Kelly, Ph.D.Vice President, Biopharmaceutical Development

KBI Biopharma, Inc.

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


• 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

• Aggregation, fragmentation, oxidation, deamidation, disulfide exchange, isomerization

» What is critical to maintain potency?


• Sizes ranging from a few kDa to >500 kDa• Secondary and Tertiary structure ranging from

relatively unordered to complex, highly ordered, multimeric states

• Glycosylation state ranging from un-glycosylated to complex, highly glycosylated states

• 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?

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

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

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

• 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

Use of Biophysical Techniques• Biophysical Techniques

• Differential Scanning Calorimetry / Microcalorimetry (DSC)» Thermal stability

• Circular Dichroism (CD)» Conformational stability – secondary structure

• Fourier Transform Infrared Spectroscopy (FTIR)» Conformational stability – secondary structure

• Fluorescence Spectroscopy» Conformational stability – tertiary structure

• Dynamic Light Scattering (DLS)» Physical stability

• Scout the various techniques to determine which are most informative for your protein

• Will depend on the unique structural features of the protein

Protein Preformulation Workflow• “Research Phase”

• Biophysical Screening

• Solubility Evaluation

• DOE & Accelerated Stability

• Forced Degradation

Identify critical factors, Eliminate non-critical factors

Preformulation “Research Phase”• Background Information

• pI• MW• Glycosylation state• Secondary & Tertiary Structure• Mechanism of Action, particularly as relates to structure

• Desired Formulation Type• Liquid, Lyophilized Powder, Other

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

Preformulation “Research Phase”

• 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)

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

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• FTIR: Secondary structure evaluation• Circular Dicroism: Secondary structure evaluation• Fluorescence: Conformational stability

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

CD and FTIR – Structural Analysis

•Overlay spectra from various candidate formulations for comparative evaluation, look for changes to core secondary structural elements

•CD more sensitive for α–helix, FTIR more sensitive for β-sheet

•Not Quantitative

-0.004

-0.003

-0.002

-0.001

0

0.001

0.002

160016201640166016801700

Wavenumber (cm-1)

AU

α-helix β-sheet

CD FTIR

CD method conditions; Structural analysis

• Buffer subtraction• Normalization (using protein concentration)• May be used to estimate secondary structural contents

Data analysis

Five (averaged)Scans/sample

100nm/minScan speed

1nmData Pitch

1 secResponse

1nmBandwidth

Jasco J-810 CD SpectrapolarimeterInstrument

100mdegSensitivity

50µLVolume requirement

0.1mm (quartz cuvette)Pathlength

190-250nm (far UV region)250-350nm (near UV region)

Scan range

2mg/mL (may be changed to obtain sufficient signal-to-noise ratio)Sample concentration

CD method conditions; T-melt analysis

20ºCStart temperature

100ºCFinal temperature

• Buffer subtraction (if available)• First derivative analysis to obtain Tm value

Data analysis

1ºC/minTemperature slope

1nmData Pitch

1 secResponse

1nmBandwidth

Jasco J-810 CD SpectrapolarimeterInstrument

100mdegSensitivity

300µLVolume requirement

1mm (quartz cuvette)Pathlength

Selected based on protein’s secondary structural content (suggested values: 222nm for proteins with predominant α-helical structure, 218nm for proteins with predominant β-sheet structure)Monitor Wavelength


FTIR method conditions

• 100µL for ATR stage• 10µL for Bio-Cell

Volume

400 (acquisition time ~20 minutes)Scans/sample

Single BeamData Type

ABB FTLA2000 FTIR (using either SensIR ATR stage or Bio-Cell)Instrument

• Background subtraction• Buffer subtraction• Vapor subtraction• Normalization• Second derivative

Data Analysis

ZeroInitial delay

4cm-1Resolution

4000-500 cm-1Scan range


Use of CD Thermal Analysis for Buffer/pH Selection

-1

3

0

1

2

CD[mdeg]

400

415

405

410

25 7530 40 50 60 70

HT[V]

Temperature [C]

-0.4

1

0

0.5

CD[mdeg]

382

390

384

386

388

25 8040 60

HT[V]

Temperature [C]

Enzyme in Acetate, Sucrose, pH 4.0 Enzyme in Tris, Sucrose, pH 8.0

Tm: 39.5oC Tm: 52.5oC

Difference of 13°C!

Structural Content by CD and FTIR

-8E+006

4E+006

-5E+006

0

200 250210 220 230 240

Mol. Ellip.

Wavelength [nm]

-8E+006

4E+006

-5E+006

0

200 250210 220 230 240

Mol. Ellip.

Wavelength [nm]

- Formulation 1

- Formulation 2

- Formulation 3

45%

45%

45%

α-helix

FTIR

35%

37%

38%

α-helix

CDβ-sheet

14%Formulation 3

13%Formulation 2

12%Formulation 1

-Lot 1

-Lot 2

-Lot 3

Amide I

Amide II

DSC method conditions

400 µL of 2mg/mL in formulation buffer (may be changed to obtain acceptable signal-to-noise ratio)Sample requirements

35ºCFill temperature

MicroCal, VP-DSC Capillary Cell MicrocalorimeterInstrument

• Buffer subtraction and normalization (using protein conc.)• Determine Tm values (mid-point of major peaks)Data analysis

NoneFeedback mode/gain

16 secFiltering period

25ºCPost-cycle thermostat

0 minPost-scan thermostat

10 minPre-scan thermostat

60ºC/hrScan rate

110ºCFinal temperature

20ºCStart temperature

Comparison of DSC and CD for Thermal Analysis

-1

4

0123

CD[mdeg]

360

374

365

370

30 8040 50 60 70

HT[V]

Temperature [C]

40 50 60 70

0

20

40

60

80

100 Data: m072606002dsc_cpModel: MN2StateChi^2/DoF = 1.674E6Tm 52.33 ±0.011∆H 4.112E5 ±2.22E3∆Hv 1.828E5 ±1.23E3

m072606002dsc_cp M072606002DSC_CPPEAK1

Cp

(kca

l/mol

e/o C

)

Temperature (oC)

Tm = 52°COnset of unfolding = 43°C


Enzyme A

Comparison of DSC and CD for Thermal Analysis

40 50 60 70

0

50

100

150

Data: m072606003dsc_cpModel: MN2StateChi^2/DoF = 6.379E6Tm1 55.37 ±0.015∆H1 4.868E5 ±7.21E3∆Hv1 2.686E5 3.17E3Tm2 59.83 ±0.13∆H2 1.350E5 ±8.19E3∆Hv2 1.733E5 1.27E4 m072606003dsc_cp

M072606003DSC_CPFIT M072606003DSC_CPPEAK1 M072606003DSC_CPPEAK2

Cp

(kca

l/mol

e/o C

)

Temperature (oC)

-1

4

0123

CD[mdeg]

370

400

380

390

30 8040 50 60 70

HT[V]

Temperature [C]


Tm1 = 55°COnset of unfolding = 47°CTm2 = 60°C

Enzyme B

Effects of pH for Glycoprotein A

20 40 60 80 100 120-0.00015

-0.00010

-0.00005

0.00000

20mM Glutamate, pH 4.0, Tm 53.2°C 20mM Glutamate, pH 4.5, Tm 57.3°C

Cp(

cal/o C

)

Temperature (oC)

Effects of pH for Glycoprotein A

20 40 60 80 100 120-0.00015

-0.00010

-0.00005

0.00000

0.00005 20mM Acetate, pH 4.5; Tm 57.1°C 20mM Acetate, pH 5.5; Tm 59.4°C

Cp(

cal/o C

)

Temperature (oC)

20 40 60 80 100 120-0.00015

-0.00010

-0.00005

0.00000

0.00005

20mM Histidine, pH 5.5, Tm 58.5°C 20mM Histidine, pH 6.5, Tm 60.1°C

Cp(

cal/o C

)

Temperature (oC)

Effects of buffer type for Glycoprotein A

20 40 60 80 100 120-0.00015

-0.00010

-0.00005

0.00000 20mM Glutamate, pH 4, Tm 53.2°C 20mM Succinate, pH 4, Tm 54.5°C 20mM Lactate, pH 4, Tm 53.3°C 20mM Glycolate, pH 4, Tm 53.8°C

Cp(

cal/o C

)

Temperature (oC)

20 40 60 80 100 120-0.00015

-0.00010

-0.00005

0.00000

0.00005

20mM Phosphate, pH 5.5, Tm 61.5°C 20mM Histidine, pH 5.5, Tm 60.1°C

Cp(

cal/o C

)

Temperature (oC)

DSC can be used to compare different lots

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

-25

-20

-15

-10

-5

0

5

10

Lot A, Replicate 1 Lot B, Replicate 2 Lot C, Replicate 3

Cp

(kca

l/mol

e/o C

)

Temperature (oC)

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

-25

-20

-15

-10

-5

0

5


Cp

(kca

l/mol

e/o C

)

Temperature (oC)

•Very similar Tm values obtained for the three lots20 30 40 50 60 70 80 90 100 110 120

-35

-30

-25

-20

-15

-10

-5

0

5


Cp

(kca

l/mol

e/o C

)

Temperature (oC)

S ample L ot T m Av g . T m S td. Dev . % R S D

L ot A, R ep 1 67.3L ot A, R ep 2 67.0L ot A, R ep 3 66.9L ot B , R ep 1 66.6L ot B , R ep 2 66.6L ot B , R ep 3 66.7L ot C , R ep 1 67.8L ot C , R ep 2 67.6L ot C , R ep 3 67.7

Av e rag e 67.1S td. Dev . 0.5%R S D 0.8

67.7

% R S D = 100*( S td. Dev. /Avg)

0.2

0.0

0.1

67.1

66.6

0.3

0.1

0.2

DLS method conditions

Sarstedt, Cat. No. 67.754 (disposable; volume requirement: 1mL)Cuvette

Automatic (selected by the instrument to optimize signal-to-noise)Attenuation

173º (backscatter)Measurement Angle

1.450 (typical for protein samples)1.330 (for water as the dispersant)Refractive Index

Malvern Zetasizer Nano-ZS, Model 3600Instrument

• Based on general purpose method (normal resolution)• Compare Z-average diameter, PDI values for overall cumulant analysis• Compare width and hydrodynamic diameter for all peaks in the intensity distribution profiles• Not recommended to be used for quantifying the relative amounts of various size species in a

sample

Data Analysis

0.8872cP (for water under these conditions)Viscosity

25°CTemperature

Five measurements averaged, each with 10-15 runsMeasurements

2mg/mL in formulation bufferSample concentration

Effect of Buffer Type for Glycoprotein A

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Size Distribution by Intensity

Record 118: 20mM phosphate, pH 6.5 Record 119: 20mM histidine, pH 6.5

Effect of pH for Glycoprotein B

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)


Record 123: 20mM succinate, pH 4.0 Record 124: 20mM succinate, pH 4.5

Effect of Buffer Type and pH for Glycoprotein B

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)


Record 125: 20mM acetate, pH 4.5 Record 126: 20mM lactate, pH 4.0Record 127: 20mM lactate, pH 4.5

Summary of DSC and DLS Screening for Glycoprotein A

Tm versus buffer type at various pH conditions

48

50

52

54

56

58

60

62

64

Glutam

ateSucc

inateLac

tic A

cid

Glycolic

Acid

Glutam

ateSucc

inateLac

tic A

cid

Glycolic

Acid

Acetat

e Citr

ate

Acetat

e Hist

idinePhosp

hate

Histidine

Citrate

Phospha

te Tris

Buffer type

Tm (d

eg C

)

Tm

pH 4.0

pH 4.5 pH 5.5pH 6.5 pH 7.0 pH 8.0

PDI versus buffer type at various pH conditions

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Glutam

ateSucc

inateLac

tic A

cidGlyc

olic A

cidGlut

amate

Succinate

Lactic

Acid

Glycolic

Acid

Acetat

e Citr

ate

Acetat

e Hist

idinePhosp

hate

Histidine

Citrate

Phospha

te Tris

Buffer type

PDI

pH 4.0 pH 4.5 pH 5.5pH 6.5 pH 7.0 pH 8.0

• DSC indicates thermal stability is a function of pH• DLS reveals differences in physical stability as a function of buffer type and pH• Chose to further evaluate Acetate, Histidine and Glycolate

Fluorescence Spectroscopy using ANS• 1,8-ANS - Amphiphilic probe that exhibits negligible

fluorescence in water yet reveals a significant increase in fluorescent intensity when bound to hydrophobic regions on a protein

• As a protein unfolds, hydrophobic core regions that are inaccessible to the dye in the native structure are exposed and bound by the dye thereby increasing the fluorescent intensity of the sample.

Fluorescence Spectroscopy using ANS

Effect of Buffer, pH and Sucrose Concentration after 11 Days at 40°C/75%RH

0

50000

100000

150000

200000

250000

300000

400 500 600 700

Wavelength (nm)

Inte

nsity

(cps

)

Tris High Sucrose pH 8

Tris Low Sucrose pH 8

HEPES High Sucrose pH 7.5

HEPES Low Sucrose pH 7.5

Fluorescence Spectroscopy using ANS

Potentially “Platform-able” Analytical Techniques

• cIEF – Imaged CE• SDS-CGE and Microchip electrophoresis

• Take advantage of manufacturer’s kit chemistry to rapidly develop preliminary methods

• SEC-HPLC• SDS-PAGE• HIAC (non-USP, low volume syringes)

• Surfactant evaluation studies» Agitation and Freeze-thaw in the absence of surfactant and at

different surfactant levels» Use in combination with DLS, SDS-PAGE, SEC

Conclusions• Biophysical techniques may be applied to a broad range of

protein therapeutics using standard analysis parameters• Use to rapidly evaluate the impact of primary formulation factors

on thermal, conformational, and physical stability • Identify a smaller subset of conditions which appear to be optimal• Characterize a preliminary design space within which the native

three dimensional conformation of the protein is maintained• May constitute part of a “platform approach” for initial

preformulation studies, greatly increasing our knowledge about the protein while stability-indicating analytical methods are still under development

Acknowledgements• Pooja Arora, PhD, Group Leader, Biopharmaceutical Development

• Juan Davagnino, PhD, Associate Director, Biopharmaceutical Development

• Vickie Dowling, PhD, Group Leader, Biopharmaceutical Development

• Wayne Yount, PhD, Group Leader, Biopharmaceutical Development

• Steven Cottle, MS, Scientist I, Biopharmaceutical Development

Thank You!

Platform Techniques for Preformulation Development for Non-Antibody Products

Health & Medicine