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UTILIZATION OF BIOPHYSICAL TOOLS TO

CHARACTERIZE VACCINES

Lakshmi Khandke, PhD Formulation Development

Vaccines Research, Pfizer Global R&D

Short Course: Challenges and Strategies in Development of Vaccines

AAPS National Biotech Conference

June 7, 2015

Overview of presentation

• Introduction to vaccines and adjuvants

• Focus of discussion – Tools to evaluate Antigen-Adjuvant interactions

• Challenges with adjuvants

• Key points

– How do we apply biophysical tools to make decisions at a pre-formulation

stage?

• What is the optimal solution condition to ensure stability of tertiary and

secondary structure?

• What are the physical parameters that affect formulations?

• Understanding adsorption to aluminum salts

• Adjuvant – Antigen interactions

• Excipient – surfactant interactions

Pfizer Confidential │ 2

How are vaccines different?

• Critical difference between vaccines and therapeutics is the lack of any activity in

vaccine antigens

– Vaccine antigens are designed to have no in vitro measurable activity

• Need for adjuvants to boost immune response

• Vaccines do not affect the body

– Efficacy is dependent upon the body responding to them

• In vitro potency assays can not measure any intrinsic activity of vaccine components

– Therapeutics, including mAb, have measurable activities including binding affinity, receptor

activation, etc.

Traditionally approach “The process of producing the vaccine defines the product”

Today’s approach “Well characterized Vaccines”


What are adjuvants?

Delivery Systems

• ISCOMS

• Virosomes

• Liposomes

• VLP

• ISCOMS

Depot effect

• Aluminum salts

• Oil in water Emulsions

• Nanoparticles

Immune modulators

• Bacterial and viral components

• Saponins

• MPL

• CpG


• Adjuvants are components added to the vaccine formulation to

enhance or modulate the immune response

Adjuvants

Safe

Stable

Co-formulated with Antigens

Combined prior to immunization

• Improve the quality and quantity

•Antigen presentation

• Influence the magnitude and avidity via mechanisms including increased antigen presentation, uptake, distribution and selective targeting

• Increase the total antibody titer or functional titers

• Increase the speed and duration of the vaccine-specific protective response

• Stabilize epitope conformation

•Dose sparing effect for the antigen

•Depot effect – slow release of antigen

•Decrease the number of immunizations needed

•Overcome competition in combination vaccines

Enhance immune responses in the young or older populations by in immature or senescent individuals

5

Immune

response

Dose

Population

Why do we need Adjuvants?

Challenges with vaccine adjuvant formulations


Compatibility with antigens

Conformational changes of

Antigen

Analytical challenges

Regulatory concerns

•Physical characteristics such as density, viscosity, pH, size and size distribution, surface charge, e.g. adsorption, binding or coupling of an antigen •Biochemical characteristics (Oxidation/deamidation/Aggregation). •Short and longer term stability

•Surfactants can change conformational epitopes leading to decreased in vitro reactivity to monoclonals •Lipid components can alter hydrophobicity

•Tight interaction between adjuvant and antigen •Difficult to quantitate individual components •Difficult to characterize antigen in the presence of adjuvants

Adjuvants are not approved by themselves but in combination with the antigen Need to generate safety database in healthy population and target age group (Prophylactic versus Therapeutic)

Adjuvants in human use

• Approved adjuvants

– Aluminum salts

– Novartis - MF59 (Flu vaccine Fluad(r)

– GSK AS04 (combination of aluminum and MPL) for viral vaccines (hepatitis B, HPV).

• Adjuvants in development and or clinical testing

– Mineral salts - e.g.,, AlPO4, Al(OH)3 and Ca3(PO4)2

– Oil emulsions such as MF59, AS02, Montanide

– Particulate adjuvants - e.g., virosomes, ISCOMS (structured complex of saponins and lipids);

ASO4;

– Microbial derivatives - e.g., MPL(TM) (monophosphoryl lipid A), CpG motifs, modified toxins

(LT and CT); AGP’s (RC529)

– Plant derivatives - e.g., saponins (QS-21);

– Endogenous immuno stimulatory adjuvants - e.g., cytokines.(hmGM-CSF, hIL12)

– Inert particles such as gold particles


Biophysical Techniques during pre-formulation

Physical

attributes

Surface Charge

Viscosity

Density

Osmolality

Turbidity

Spectroscopy

Secondary

structure (far-UV

CD, FTIR, Raman)

Tertiary structure

(near-UV,

Fluorescence,

NMR, XRD)

Thermal Analysis

Thermostability,

Protein Structure

(VP-DSC)

Ligand binding

(Titratration

Calorimetry)

Lyo powder and

frozen sol.

characterization,

i.e Tg, Tg’,

Melting,

Crystallization,

Enthalpy

Relaxation

(Modulated DSC)

AUC*

Absolute

Mass

Detect

aggregates

LS

“Classical”

Molecular

weight, Aggs

OD 350

Nephelometry

“Dynamic”

hydrodynamic

size

precipitation

*Analytical Ultracentrifugation

Why do we need Biophysical characterization in

vaccine drug product development?

• Secondary and tertiary structure of molecule

• Optimizing solution conditions

• Excipients • pH • Stabilizers

• Thermal stability • Predict solution

stability • Physical properties

• Conformational

stability • Secondary tertiary

structure • Mechanism of

interaction/ binding • Physical parameters

such as particle size, surface charge, viscosity

Demonstrate enhance

immune response in vivo

• Process Design • Consistency of

manufacture • Physical properties

DEMONSTRATE STABILITY

Antigen

Adjuvant Antigen

Process

LIQ LYO

9

How do you monitor stability of vaccine with an

adjuvant?

• Example 1

– Combination of antigen-adjuvant prior to immunization


Parameters used to assess stability for a lyophilized drug product

reconstituted with an adjuvant as a function of time

In addition: Biophysical tools to characterize the interaction during pre-formulation

Questions –

• Does the conformation remain the same

• Do they interact with each other?

• Is it important?


Physical characteristics • Recon Time • Appearance • pH • Osmolality

Antigen • Concentration /Purity - HPLC assays - AEX – strength, purity, deamidation, oxidation etc - RP-HPLC - SEC aggregation

Adjuvant stability • concentration / purity

• HPLC based assays • Particle size

TOOLS Circular Dichroism

Secondary structure characterization – far UV (190-250 nm)

Tertiary structure characterization – near UV (250-350 nm)

Fluorescence - Intrinsic (Trp) structure characterization

DSC – Tertiary structure

ITC - Thermodynamic interaction between protein(s) and adjuvant

Case study: Bi-valent vaccine (lyophilized)

recon with Adjuvant


Optimal stability of (Lyo DP) 2 antigens

Stable pH 7.4

Optimal stability of Adjuvant

pH 5.3 – 5.6

Combined pH 6.3 to 6.6

Recovery and purity should be within target for strength and

purity for Adjuvant and Antigen using HPLC

based assays Tertiary and secondary structure

using Biophysical tools Fluorescence, CD, DSC, AUC, ITC

Combined antigen/adjuvant is stable within a

narrow pH range

Signal Antigens pH 5.0 pH 6.0 pH 7.0 pH 8.0

DSC Tm (°C) A 47.9 48.8 49.3 47.0

B 40.2 48.7 50.9 50.0

Fluor. Tm (°C) A 38.8 46.3 41.3 38.8

B 36.3 36.3 38.8 36.3

CD Tm (°C) A 41.1 46.3 48.7 67.8

B 49.1 50.7 53.4 53.1


Key points:

1. Antigen: Higher the Tm better stability

• Lower pH prone to aggregation (based

on traditional accelerated stability)

2. Adjuvant: Low pH more stability

3. Combination: Antigen and adjuvant strength

and purity is maintained for up to six hours

providing a narrow window for dosage

delivery

Biophysical analyses of Antigens

Adjuvant stability in the presence of antigens

0

20

40

60

80

100

120

T0 0.5 hr 2 hrs 4 hrs 6 hrs 24 hrs

% R

eco

very

of

Ad

juva

nt

Post- Reconstitution Time

pH 5.3 pH 5.6 pH 5.9

No change in 2°/3° structure of proteins studied in the presence of adjuvant

Far UV CD No change in secondary structure of Protein A/B

Protein A+B + Adjuvant Protein A+B

Near UV CD No change in tertiary structure of Protein A/B

Protein A/B + Adjuvant Protein A/B

Sample Avg. Tm1

(n=3) SD

(n=3)

Protein A 47.45 0.10

Protein A + Adjuvant 47.52 0.11

Protein B 51.50 0.32

Protein B + Adjuvant 51.35 0.21

DSC No change in Tm1 of protein in presence of adjuvant

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

10 20 30 40 50 60 70 80 90 100

Antigen with Adjuvant

Antigen Alone

Both Antigen 1 and 2 are thermostable over a pH range of 6.0 to 7.0

in the presence of the adjuvant

Antigen A Antigen B Trp Fluorescence No change in tertiary structure of protein in presence of adjuvant

No interaction between protein and adjuvant studied using – ITC and AUC

ITC AUC

• Adjuvant does not change Protein A/B size distribution

• No adjuvant binding can be detected by AUC

• No difference in ITC profile with and without adjuvant

Protein B + Adjuvant Protein B Buffer - Buffer

Protein B Buffer Protein B Adjuvant

Protein A + Adjuvant Protein A

Buffer - Buffer Protein A Buffer Protein A Adjuvant

Summary

• Stability of the adjuvant and antigen may not be the same and

hence co-formulation can be challenging to obtain long term

stability

• Adjuvants used to reconstitute vaccine prior to immunization

• Structural conformation can be obtained in the presence of

adjuvants

• Interaction between the Ag and Adjuvant may be important in

certain cases


Aluminum containing vaccines


Important attributes Biophysical tools

Adsorption to aluminum salts Binding isotherms Optimizing excipients/pH Lot to lot consistency Structural integrity

Physical parameters • Particle size – Malvern, MFI • Charge – Zeta potential • Turbidity / Settling rates • Viscosity/ Surface tension • Break loose and extrusion

forces

Immune response Long term stability

Iso thermal calorimetry Front face fluorescence Differential scanning calorimetry FTIR

Process Development Mixing/Settling

Filling uniformity Pre-filled syringes

Al

Ag

Salt Buffer

pH

Aluminum salts


Aluminum Hydroxide Adjuv.

Crystalline

Primary particles: fibers

Surface OH groups

IEP = 11.4

+ surface charge at pH 7.4

Aluminum Phosphate Adjuv.

Amorphous

Primary particles: plates

Surface OH and PO4 groups

IEP = 4-6

- surface charge at pH 7.4

Stanley Hem

AH AP

Mechanism of adsorption to aluminum

Mechanism of binding

Hydrophobicity

Surface charge

Ligand exchange

Steric

factors


Polysaccharide (PnP) Proteins PnC

Charge, hydrophobicity/hydrophilicity Multiple conformation due to conjugation chemistries.

Surface Accessibility of each component, Conformational flexibility

AlPO4

Surface charge of the antigen and aluminum are

important for adsorption


-30

-20

-10

0

10

20

30

40

5.2 5.6 6.0 6.5 7.0 7.3 7.6 7.9

(m

V)

Zeta potential of Al(OH)3

R² = 0.9956

-14

-12

-10

-8

-6

-4

-2

0

6.2 6.4 7.2 7.8

mV

pH

Antigen A: pI = 4.3

Antigen B: pI = 5.3

Optimal pH for proteins is 7.0 - 8.0

Formulation pH chosen : 7.4

Complete binding of antigens to aluminum

How much aluminum is required to adsorb the antigens

Can be determined by adsorption capacity & coefficient

• Adsorption is described by two parameters –

o The maximum amount that can be adsorbed as a monolayer – CAPACITY

o The strength of adsorption force – COEFFICIENT

• Linear Langmuir equation (derived from an adsorption isotherm) 1 is used to describe adsorption, in which the solute is adsorbed to form a monolayer

21

𝑐

𝑦=

𝑐

𝑦𝑚+

1

𝑏𝑦𝑚

c : concentration of the protein in solution y : mass of protein adsorbed per mass of adjuvant

b : adsorption coefficient ym: adsorption capacity

1Hansen B, Belfast M, Soung G, Song L, Egan PM, Capen R, Hogenesch H, Mancinelli R, Hem SL. Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response. Vaccine. 2009 Feb 5;27(6):888-92

• To generate the adsorption isotherms and linear Langmuir plots -

o Add varying protein concentrations to adjuvant (e.g. Alhydrogel®) suspensions

o Post incubation, determine c and y using UV280

o A typical adsorption isotherm is shown below, with three regions 2

22

Protein in solution, c

Pro

tein

ad

sorb

ed p

er m

g o

f ad

juva

nt,

y

Rate of increase in adsorption related to adsorption coefficient

Constant adsorption indicates formation of monolayer, related to adsorption capacity

Multilayer adsorption

2Jendrek S, Little SF, Hem S, Mitra G, Giardina S. Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants. Vaccine. 2003 Jun 20;21(21-22):3011-8

Background – Adsorption capacity & coefficient

• To determine capacity and coefficient 2

o Data from adsorption isotherm (e.g. monolayer – first few concentrations) used

o Plotted according to the linear Langmuir equation

o Determine b (coefficient) as slope/intercept and ym (capacity) as 1/slope

23


Pro

tein

ad

sorb

ed p

er m

g o

f ad

juva

nt,

y


Pro

tein

in s

olu

tio

n/

Pro

tein

ad

sorb

ed, c

/y

ym =1

𝑆𝑙𝑜𝑝𝑒

b =𝑆𝑙𝑜𝑝𝑒

𝐼𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

Concentrations used to generate linear Langmuir plot

2Jendrek S, Little SF, Hem S, Mitra G, Giardina S. Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants. Vaccine. 2003 Jun 20;21(21-22):3011-8

Background – adsorption capacity & coefficient

Understanding of adsorption can help develop formulations

24

Addition of phosphate buffer can influence the tightness of binding to aluminum

Weaker interactions help increase desorption of the antigens

Protein Phosphate in 1 mg/mL

Alhydrogel® (mM) Adsorption Coefficient

(ml/mg)

Adsorption Capacity (mg protein/ mg Al)

A 0 121.5 4.16

25 6.7 0.69

B 0 153.0 3.52

25 8.5 0.42

How did we employ Biophysical tools for a

conjugates during pre-formulation?

• Determine the tertiary structure of glyco

conjugates in relation to CRM197

– Does the conjugation process affect the

structure?

– Is there a difference between lots?

• Rationale for pH selection and optimizing

aluminum levels formulation help determine a

sweet spot for maximizing binding and product

stability

• Understand the impact of adsorption to

aluminum salts on structure and stability of the

carrier protein CRM197.

– Does binding to aluminum unfold the

protein given that unfolding may lead to

instability?


CRM197

Carrier

Polysaccharide-CRM197 Conjugate

(Complex cross-linked conjugate)

Polysaccharide

Front Face Fluorescence

- peak shift of intrinsic fluorescence upon adsorption to Al(OH)3 and AlPO4 adjuvants


The antigens are completely bound to aluminum

Upon binding of the protein to aluminum there is a conformational shift

AlPO4 better maintains protein tertiary structure than hydroxide upon protein adsorption

With Alhydrogel there is a delayed onset of transition, however it is known that the tightness of binding

increases what may or may not be desired depending upon the antigen

Glyco conjugates have varied binding capacity to aluminum based

on binding isotherms


Level of cross linking may influence

accessibility of CRM protein

Formulation pH is a balance between stability and

adsorption to aluminum


-1000

0

1000

2000

3000

4000

5000

20 25 30 35 40 45 50 55 60

Cp (k

cal/

mol

e/C)

Temperature (C)

pH Effect on the Transition of Serotype 1 by VP-DSC

pH 5.00

pH 5.25

pH 5.50

pH 5.75

pH 6.00

pH 6.25

pH 6.50

pH 6.75

pH 7.00

30

32

34

36

38

40

42

44

5 6 7

TmC

PH

0

20000

40000

60000

80000

100000

120000

4.50 5.00 5.50 6.00 6.50 7.00

Flu

ore

sce

nce

In

ten

sity

pH

0

20

40

60

80

100

120

A B C D E

% A

g ad

sorb

ed

to

Al

Example conjugates

pH 5.2

pH 5.5

pH 5.8

pH 6.1

pH 6.4

Decreasing pH leads to decreased stability

Increasing pH leads to decreasing binding

Representative

Glyco conjugate

Excipient –surfactant interactions

• Example -3

– Prefilled syringes and surfactants


30

Silicone in prefilled syringes can lead to

product aggregation

Product

Agitation

Product

Particulates

Silicone

Siliconized HYPAK syringes Un-siliconized syringes

-10

0

10

20

30

40

50

C E G F H A B D L I M J K

To

tal A

nti

ge

nic

ity

lo

ss

(%

)

2 hrs agitation

8 hrs agitation

24 hrs agitation

Best case

-10

0

10

20

30

40

50

C E G F H A B D L I M J K

% a

nti

gen

lo

st

2 hrs agitation

8 hrs agitation

24 hrs agitation

Worst case

Simple model of the interfacial behavior of

surfactant

31

Hydrophilic groups are oriented toward the bulk water and the hydrocarbon chains (tail) are

pointed towards the air or hydrophobic solid

At or above CMC, there is an oriented monolayer of surfactant molecules and maximum

surfactant absorption

Silicone Oil in Pre-filled syringe (PFS)

Silicone PS80

32

0.00 0.05 0.10 0.15 0.20 0.25

-0.04

-0.03

-0.02

-0.01

0.00

0.01

0.02

0.03

0.04

0.05-0.04

-0.03

-0.02

-0.01

0.00

0.01

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µca

l/se

c

Molar Ratio

kca

l/mo

le o

f in

ject

ant

Glyco conjugates are stabilized by Polysorbate-80 through

competition with the surface

ITC Example: Conjugate in 0.02% PS80 – Manual ITC

–No interaction

Typical example of ITC with stepwise injection to

measure the heat of interaction

•PS-80 is required for PnC stability in prefilled syringes, which contain silicone oil for syringe functionality. •PS-80 usually stabilizes the proteins either through interaction with proteins directly in solution or competition with the proteins for the interface. •No interactions between PS-80 and the conjugates. PS 80 interacts with the glass surface and silicone oil, stabilizing the conjugates from damage due to interfacial stress.

Utility of particle size measurements

• Example – 4

– Use of particle size analyses in formulation and process

development



Particle size can be used for process development to determine

limits of mixing speed / filling prefilled syringes

Excess Mixing

Increases

shear

Decrease in particle size

Decrease in settling time

Denser settlement of Al

at the tip of a syringe leading to increased resuspension times

0

10

20

30

40

50

60

R0 R8 R16

No

. of

inve

rsio

ns

Recirculation Times

Resuspendability 1month 5C

1 month 37C

Particle size of aluminum increases on freezing


0

5

10

15

20

25

30

35

40

45

50

D10 D50 D90

Par

ticl

e s

ize

(u

m)

Control 1X F/T

0

2

4

6

8

10

12

0.11 0.15 0.19 0.24 0.31 0.41 0.52 0.68 0.87 1.13 1.45 1.88 2.42 3.12 4.03 5.21 6.72 8.68 11.2 14.5 18.7 24.1 31.1 40.1 51.8 66.9 86.4

Par

ticl

e s

ize

(u

m)

Pre-shipping

shipped

Non-shippedcontrol

Particle size measurement using Malvern analyzer can be useful to determine stability of aluminum

particles following a shipping excursion

-20C

When do we use what tools?

Phase Tools

Pre-formulation

Selection of optimal pH DSC, Fluorescence, CD, OD 350

Antigen-Antigen/excipients/Adjuvant interactions

ITC

Antigen-Adjuvant in combination DSC, Fluorescence, CD, AUC, particle size (DLS/ MFI), surface charge

Vaccines with aluminum salts

Adsorption to Al salts Surface charge, Binding isotherms

Stability on Al surface Front face fluorescence

Formulation and process development Surface charge, surface tension, viscosity, particle size (Mastersizer), immuno assays such as Nephelometry or ELISA based


Biophysical techniques can be used as a characterization tools in

vaccine pre-formulation development

Optimize formulation

Mechanism of interaction/ adsorption

Physical parameters such as particle size,

surface charge, viscosity

Conformational stability of antigen

Formulation

Optimization

Process Development

Stability

Interplay Secondary and

tertiary structure


Acknowledgements

Pfizer Colleagues

• Amardeep Bhalla

• Cindy Yang

• Kunal Bakshi

• Ozgur Akcan

• Leena Bagle

• Oleg Jouravlev

• Karen Xu

• Lynn Phelan

• Mark Ruppen

• Kathrin Jansen

38

UTILIZATION OF BIOPHYSICAL TOOLS TO

CHARACTERIZE VACCINES

Lakshmi Khandke, PhD Formulation Development

Vaccines Research, Pfizer Global R&D

Short Course: Challenges and Strategies in Development of Vaccines

AAPS National Biotech Conference

June 7, 2015

lakshmi khandke, phd - zerista.s3.amazonaws.com · i.e tg, tg’, melting, crystallization,...

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