Protein Stabilization and Delivery: A Case ... - gea.ku.edu

Protein Stabilization and Delivery: A Case Study with Invasion Plasmid Antigen D

Nicole Montoya

Institute of Sustainable Engineering

Department of Chemical and Petroleum Engineering

University of Kansas, Lawrence

Vaccine Cold Chain

• Transporting and storing vaccines at 2-8ºC from

manufacturing to delivery site

• 50% vaccine wastage in the past 15 years

• Main problems:

• Inadequate cold chain capacity

• Lack of functioning cold chain equipment

• Poor temperature monitoring and

maintenance systems

WHO/UNICEF Achieving immunization targets with the comprehensive effective vaccine management (EVM) framework.

Ultimate Goal

Desorbing

Agent

Mem

bra

ne

Mem

bra

ne

Silica Immobilized Vaccine

Mem

bra

ne

Mem

bra

ne

Silica + Desorbing Agent

Desorbing agent is pushed

through membrane

Free Vaccine

Vaccine diffuses through

membrane Vaccine is now ready for

administration

To develop and manufacture a syringe-like device that transports and

stores silica immobilized vaccine

Background

Shigellosis & IpaD

• Shigellosis is a gastrointestinal disease (1 million deaths annually)

• Invasion Plasmid Antigen D (IpaD): antigen protein → target protein for stabilization

Mesoporous Silica & IpaD

Physicochemical Characterization (BET)

Silica GelAverage Pore Diameter

(nm)

Accessible Pore Volume

(cm3/g)

Accessible Surface Area

(m2/g)

Sample 1 (S1) 3.9 ± 1.5 0.07 ± 0.01 67.4 ± 3.3

Sample 2 (S2) 8.1 ± 1.5 0.30 ± 0.01 125.6 ± 3.3

Sample 3 (S3) 15.0 ± 1.5 1.28 ± 0.01 333.2 ± 3.3

Sample 4 (S4) 17.7 ± 1.5 1.41 ± 0.01 319.3 ± 3.3

Sample 5 (S5) 24.2 ± 1.5 2.18 ± 0.01 370.7 ± 3.3

Sample 6 (S6) 30.2 ± 1.5 3.78 ± 0.01 300.0 ± 3.3

Sample 7 (S7) 36.3 ± 1.5 2.87 ± 0.01 286.1 ± 3.3

Langmuir 2020, XXXX, XXX, XXX-XXX Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Materials and Methods

Adsorption Washing Heating DesorptionCircularDichroism

• 30 mg silica gel + 0.7 mL IpaD at 1.5 mg/mL

• Mix for 20 hours

• Measure supernatant protein concentration

• Wash silica to remove unbound proteins

• Heat silica-IpaD complex at 95C for 2 hours

• Remove proteins from silica with 0.7 mL of 10% LDAO

• CD analysis on IpaD to evaluate secondary structure

LDAO Structure:

Effect of silica pore diameter on IpaD adsorption

Key Results:

• Percent IpaD adsorption increases

as pore volume increases

• Silicas with pore diameter > 15 nm

adsorb more than 90% IpaD

Pore Diameter (nm)

0 10 20 30 40

Perc

ent

Ipa

D A

dso

rbed

0

20

40

60

80

100

S4

S1

S2

S3

S6 S7S5

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Adsorption Isotherm Silica Gel S3

Langmuir Model Equation:

𝐶𝐼𝑝𝑎𝐷,𝑎𝑑𝑠 =𝐾𝐶𝑚𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒

1+𝐾𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒

Linearized Form:𝐶𝐼𝑝𝑎𝐷,𝑎𝑑𝑠

𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒=

1

𝐾𝐶𝑚+

𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒

𝐶𝑚

• Cm: maximum monolayer coverage

• K= kads/kdes

Free IpaD Concentration (mg/mL)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Adso

rbed I

pa

D (

mg

/g)

0

50

100

150

200

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Adsorption Isotherm Silica Gel S3

Cm=181 mg/g

K=21

Free IpaD Concentration (mg/mL)

0.0 0.2 0.4 0.6 0.8 1.0

Fre

e/A

dso

rbed I

pa

D C

once

ntr

ati

on (

g/m

L)

0.000

0.002

0.004

0.006

0.008

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

IpaD Unit Cell Dimensions 10.07 nm by 11.2 nm

Unit Surface Area 112.8 nm2/protein

IpaD Molecular Weight 39 kDa

181 𝑚𝑔 𝐼𝑝𝑎𝐷

𝑔 𝑠𝑖𝑙𝑖𝑐𝑎𝑥𝑚𝑜𝑙 𝐼𝑝𝑎𝐷

3.9𝑥107𝑚𝑔𝑥6.02𝑥1023 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠

1 𝑚𝑜𝑙𝑥112.8 𝑛𝑚2

𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠𝑥

(1 𝑚)2

(1𝑥109 𝑛𝑚)2=316 𝑚2 𝐼𝑝𝑎𝐷

𝑔 𝑠𝑖𝑙𝑖𝑐𝑎

Adsorption Isotherm Silica Gel S4 and S5

Silica Gel N2 Surface Area (m2/g) IpaD Surface Area (m2/g)

S3 333 ± 3.3 316 ± 8.9

S4 319 ± 3.3 317 ± 11.4

S5 371 ± 3.3 372 ± 20.2

Free IpaD Concentration (mg/mL)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Fre

e Ip

aD

/Ad

sorb

ed I

paD

Con

cen

trati

on

(g/m

L)

0.000

0.002

0.004

0.006

0.008

0.010

Free IpaD Concentration (mg/mL)

0.0 0.2 0.4 0.6

Fre

e/A

dso

rbed

Ip

aD

Con

cen

trati

on

(g/m

L)

0.000

0.001

0.002

0.003

0.004

Silica Gel S4 Silica Gel S5

Silicas with Smaller Pore Size (<15 nm)

Free IpaD Concentration (mg/mL)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Ad

sorb

ed I

pa

D C

on

cen

tra

tio

n (

mg

/g)

0

50

100

150

200

Free IpaD Concentration (mg/mL)

1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44

Adso

rbed I

paD

Conce

ntr

ati

on (

mg/g

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Silica Sample Pore Diameter

S1 (black) 3.9 nm

S3 (red) 15 nm

• IpaD cannot fit inside the pores

and adsorption is negligible

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Silicas with Larger Pore Size (>25 nm)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Free IpaD Concentration (mg/mL)

Fre

e/A

dso

rbed I

paD

Conce

ntr

ati

on (

g/m

L)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

0.000

0.002

0.004

0.006

0.008

Free IpaD Concentration (mg/mL)

Fre

e/A

dso

rbed I

paD

Conce

ntr

ati

on (

g/m

L)

294𝑚𝑔 𝐼𝑝𝑎𝐷

𝑔 𝑠𝑖𝑙𝑖𝑐𝑎𝑥

𝑚𝑜𝑙 𝐼𝑝𝑎𝐷

3.9𝑥107𝑚𝑔𝑥6.02𝑥1023 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠

1𝑚𝑜𝑙𝑥630.0 𝑛𝑚3

𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠𝑥

(1 𝑐𝑚)3

(1𝑥107 𝑛𝑚)3=

2.87 𝑐𝑚3 𝐼𝑝𝑎𝐷

𝑔 𝑠𝑖𝑙𝑖𝑐𝑎

Silica

Sample

N2 Accessible Surface

Area (m2/g)

IpaD Surface Area

m2/g

N2 Volume

(cm3/g)

IpaD Volume

(cm3/g)

SA6 300.0 ± 3.3 603.4 ± 43.5 3.78 ± 0.01 3.49 ± 0.23

SA7 286.1 ± 3.3 514.2 ± 46.8 2.87 ± 0.01 2.87 ± 0.26

• Hypothesis: Multilayer adsorption instead of monolayer adsorption

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Types of Pore Interaction with IpaD

A. Pore size less than 15 nm: IpaD does not

fit into the pore

B. Pore size between 15-25 nm: monolayer

coverage, hydrogen bonds depicted by red

line

C. Pore size larger than 25 nm: multilayer

coverage, van der Waals forces

Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020

https://doi.org/10.1021/acs.langmuir.0c02400

Circular Dichroism: Secondary Structure

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

190 200 210 220 230 240 250 260

Mo

lar

elli

pti

city

(

)

Wavelength (nm)

IpaD heated on silica

Native IpaD

Denatured IpaD

Key Results:

• Adsorbed IpaD after heat treatment

(and desorption) displays similar “W”

shaped CD signal as the native

unheated IpaD

• IpaD was heated to 95C for 150

minutes while attached to S4

Langmuir Publication

LANGMUIRpubs.acs.org/Langmuir

The ACS journal of fundamental interface science

December 1, 2020 Volume 36 Issue 47

Acknowledgements

• Dr. David Corbin

• Dr. Philip Gao

• Prof. Shiflett and Prof. Allgeier Research

Groups

• Dr. Ana Rita Morais

• Simon Velasquez Morales

• Kaylee Barr

• Rhianna Roth

• Eric Hartman

Visit our website: www.shiflettresearch.com

Thank you for you attention! Questions?