melt processing of functional protein/polymers...
TRANSCRIPT
Jon K. PokorskiUniversity of California, San Diego (UCSD)
Department of NanoEngineering
Melt Processing of Functional Protein/Polymers Blends
LAII, La Jolla, CA2/7/2020
Protein/Polymer Blends: Why do we care?Biological Pharmaceuticals
Nature Biotechnology, 1136–1145 (2018)Vaccines Enzymes
Antibodies
2017 vs 2025
Depot Protein Delivery: Emulsions
• Controllable release profiles• Protect proteins in vitro and in vivo• Micro- to nano- sized particles
Solvent Removal
Drying
Aqueous Protein Solution
OrganicPLGA
Solution
Primary Emulsion
Secondary Emulsion
Aqueous PVA
Solution
PLGA/Protein Microparticles
0 50 100 150 200 250-100
-50
0
Hea
t Flo
w (m
W/g
)
Temperature (oC)
Qβ
0 50 100 150 200-600
-400
-200
0
Hea
t Flo
w (m
W/g
)
Temperature (oC)
PLGA
Tg
Gla
ssy
Rub
bery
Viscous Liquid
Qβ VLPPLGA
• 100% Encapsulation• Inexpensive• High Loading• Scalable
Polymer Melt Processing
http://www.technologystudent.com/
Protein/Polymer Dry Blend
Challenges• High Temperature• Scale• Shear• Pressure
Protein Stability in Non-traditional Environments
Hartl, et al. Nature, 2011, 324-332.L.H.G. van Donkelaar, et al. Food Res. Int. 2015, 241–246.
Pilot-scale Melt Processing
2.35 mm3/s
1st Generation: Syringe Extruder
3rd Generation: Pneumatic Piston
Lee, P. W..;...Pokorski, J.K. Macromol. Biosci. 2015.Wirth, D.M.; Pokorski, J.K Polymer, 2019.
MonoPEGylated Lysozyme Extrusion
Lee, P.; Towslee, J.; Maia, J.; Pokorski, J. Macromol. Biosci. 2015, 1332.
4000 3500 3000 2500 2000 1500 1000
0.2
0.4
0.6
0.8
Abso
rban
ce
Wavenumber (cm-1)
Lysozyme Lysozyme-c-PEG Amide II
C-O
Amide ILysozyme-co-PEG
Lysozyme Lysozyme + PEG Lysozyme-co-PEG0.0
0.2
0.4
0.6
0.8
1.0
Frac
tion
of R
etai
ned
Activ
ity
Lysozyme
Goal: Use polymer manufacturing tocreate degradable polymer implantsto slowly release vaccine candidates
• Enhance patient compliance• Eliminate cold-chain• Ease financial-burden• Improve efficacy
Qβ as a Vaccine Platform
• VLP derived from bacteriophage Qβ• 180 copies of a single coat protein• Recombinantly expressed in high-yields• Combinatorial vaccine platform• At least 8 Qβ vaccines in clinical trials
28 nm
Repetitive Dosing
HN S
ONH
O
N
O
O N
N
Melt Processing: Qβ as a Nanofiller
2.35 mm3/s
HO
O
O
O
OH
PLGA
Qβ
Mn = 5-20 kDaHydrolytically Degradable
1-10 w/w%1 cm
Lee, P.W.; Shukla, S.; Wallat, J.D.; Danda, K.C.; Maia, J.; Steinmetz, N.F.; Pokorski, J.K. ACS Nano, 2017, 8777-8789.
Lee, P.W….Pokorski, J.K. Macromolecular Bioscience, 2015, 1332-1337.
Qβ: Pre- and Post-Processing
0 5 10 15 20 25 30 350
100
200
300
400
500
600
Abs
orba
nce
Retention Volume (mL)
280 nm 260 nm
0 20 40 60 80 100 1200
10
20
30
40
50
60
% M
ass
Radius (nm)
Pre-processed
Rh = 15.1 nm
28 nm
0 5 10 15 20 25 30 350
20
40
60
80
100
Abs
orba
nce
Retention Volume (mL)
280 nm 260 nm
0 20 40 60 80 100 1200
10
20
30
40
50
60
% M
ass
Radius (nm)
Rh1 = 12.6 nmRelative Amount = 83.8%
Rh2 = 43.2 nmRelative Amount = 16.2%
Post-processed
ACS Nano, 2017, 8777-8789.
Qβ/PLGA: Particle Distribution EDS/SEM
1% Qβ 5% Qβ 10% Qβ
• EDS specifically maps sulfur atoms from cysteineACS Nano, 2017, 8777-8789.
Multistep Processing: Nanocomposites
HO
O
O
O
OH
PLGA/1% Qβ
Compound
• Extrusion• Injection Molding• Compression Molding• Etc, etc….
• 100⁰C, 5 min, 1200 psi
0 5 10 15 20 25 30 350
50
100
150
200
250
300
Abs
orba
nce
Retention Volume (mL)
280 nm 260 nm
0 20 40 60 80 100 1200
10
20
30
40
50
60
% M
ass
Radius (nm)
Rh1 = 16.3 nmRelative Amount = 74.4%
Rh2 = 71.3 nmRelative Amount = 25.6%
Extruded and Melt PressedCompression Molding
ACS Nano, 2017, 8777-8789.
0 5 10 15 20 25 30 350.0
0.2
0.4
0.6
0.8
1.0
1.2
Rel
ativ
e A
bsor
banc
e
Retention Volume (mL)
280 nm 260 nm
0 5 10 15 20 25 30 350.0
0.2
0.4
0.6
0.8
1.0
1.2
Rel
ativ
e A
bsor
banc
e
Retention Volume (mL)
280 nm 260 nm
0 5 10 15 20 25 30 350.0
0.2
0.4
0.6
0.8
1.0
1.2
Rel
ativ
e A
bsor
banc
e
Retention Volume (mL)
280 nm 260 nm
How does shear affect Qβ?Extrusion/Injection Molding
• 95°C, 5 minute melt time, 3 minutes of applied shear
1 s-1 50 s-1
0 50 100 150 2000
10
20
30
40
% M
ass
Radius (nm)
1 s-1
0 50 100 150 2000
10
20
30
40
50
% M
ass
Radius (nm)
10 s-1
0 50 100 150 2000
10
20
30
40
% M
ass
Radius (nm)
50 s-1
5 s-1SEC
DLS
ACS Nano, 2017, 8777-8789.
VLP Stability and Peclet Number
100 101 1020
1
2
3
<R>/
<R0>
Peclet Number
0.1 1 10Shear Rate (s-1)
𝑃𝑃𝑃𝑃𝑑𝑑𝑑𝑑𝑑𝑑 =12𝜋𝜋𝜋𝜋�̇�𝛾𝑅𝑅3𝜉𝜉𝐶𝐶𝑘𝑘𝑏𝑏𝑇𝑇
η = viscosity of the polymer melt (Pa∙s)�̇�𝛾 = shear rate applied to the system (s-1)R = weight average radius of the particles pre-shear (m)T = temperature of the system (K)
• Dimensionless number analysis allows translation to other systemsACS Nano, 2017, 8777-8789.
When <R>/<Ro> = 1, Particles are Stable
Tuning Release Kinetics
1-10% w/w% Qβ
• Release profiles can be tuned by w/w% Qβ or PEG
10% w/w% PEG
PBS pH 7.4
0 10 20 30 40 50 60 70 800
20
40
60
80
100
120
% C
umul
ativ
e R
elea
sed
Days
1% Qβ/PLGA 1% Qβ/10% 8K PEG 1% Qβ/10% 20K PEG
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
% C
umul
ativ
e R
elea
sed
Days
1% Qβ 5% Qβ 10% Qβ
Biological PropertiesIgG Response to Qβ
50 µg Qβ
0 Days
10 w/w% Qβ
0 10 20 30 40 50 60 70 800
2
4
6
End-
Poin
t IgG
Tite
r (lo
g10)
Days
Injection Implant
23% 28%
65% 56%
13% 16%
Injection Implant0
20
40
60
80
100
IgG
Sub
type
Per
cent
age
IgG2b IgG2a IgG1
0 10 20 30 40 50 60 70 800
2
4
6
End-
Poin
t IgG
Tite
r (lo
g10)
Days
Injection Implant
ACS Nano, 2017, 8777-8789.