polymers and hydrogels for stabilization and delivery of animal feed enzymes and other biologics:...

Heather D. MaynardDepartment of Chemistry and Biochemistry & California NanoSystems Institute

University of California, Los Angeles

Polymers and Hydrogels for Stabilization and Delivery of Animal Feed Enzymes and

Other Biologics

Proteins are Important Therapeutic Agents

http://www.drugs.com/stats/top100/2013/salesAlconcel, et al. Polym. Chem. 2011, 2, 1442.

Leader, et al. Nat. Rev. Drug Discov. 2008, 7, 21.

Top 10 Drugs by Sales (2013)

EnbrelNeulasta

Replace deficient or abnormal proteins– diabetes, hemophilia, cystic fibrosis

Augment existing activity– neutropenia, Hep C, multiple sclerosis,

osteoporosis, sepsisNew functions or activities

– leukemia, dystonia, thrombosisTargeting/Vaccines/Diagnostics

– cancer, HIV, Lyme disease, Hep B, arthritis

Rank Drug Sales(billion USD)

1 Abilify 6.3

2 Nexium 6.0

3 Humira 5.4

4 Crestor 5.2

5 Cymbalta 5.1

6 Advair Diskus 5.0

7 Enbrel 4.6

8 Remicade 4.0

9 Copaxone 3.6

10 Neulasta 3.5

Proteins Have Formulation Challenges

Proteins are generally unstable and challenging to transport and store

At a minimum, material is discarded/wasted due to concerns over activity, and at a maximum, the drug does not make it to clinical trials due to issues with instability

Proteins require regulated temperatures and addition of tailored excipients to maintain bioactivity, yet often still have short shelf lives

Proteins Have Pharmacokinetic Challenges

Most proteins are injected and rapidly degrade in the body by natural mechanisms

In order to have a sustained affect, the patient must endure many injections

Covalent attachment of poly(ethylene glycol) (PEG) addresses these issues

Protein-Polymer Conjugate Therapeutics

• PEGylated proteins – the gold standard in protein-polymer conjugates

• 10 FDA-approved protein-polymer conjugate drugs – exclusively PEGylatedproteins

• Limitations of PEGylation– PEG accumulates in the body and some

people have an antibody response– PEG does not stabilize proteins outside

the body– PEG not tailored to specific protein– Alternatives are needed

Fishburn, C. S. J. Pharm. Sci. 2008, 97, 4167; Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. Polym. Chem., 2011, 2, 1442; Pelegri-O’Day, E.; Lin, E. –W.; Maynard, H. D. JACS, 2014, 136, 14323

Example: G-CSF-PEGStimulates production of white blood cells

Used in conjunction with chemotherapy

Maynard GroupRational Design to

Enhance Protein andSmall Molecule Drug Delivery

Hydrogels that Stabilize Proteins

Nanoparticles for Protein and Small Molecule Drug Delivery

Polymers that Borrow from Nature to Stabilize Therapeutic Proteins

Polymers that Mimic Nature to Stabilize and Enhance Activity

Nguyen, et al., Nature Chemistry, 2013, 5, 221

Lee, et al., Biomacromolecules, 2013, 14, 2561Lee, et al., Polymer Chem, 2015, 6, 3443

Matsumoto, et al., ACS Nano, 2013, 7, 867

Biomimetic polymers

Mowery, P.; et al. Synthetic glycoprotein mimics inhibit L-selectin-mediated rolling and promote L-selectin shedding. Chem. Biol. 2004, 11, 725-732.

Mimic structure

Kouwer, P. H. J; et al. Responsive biomimetic networks from polyisocyanopeptide hydrogels. Nature 2013, 493, 651-655.

Mimic biological system

Goals of this research:

Prepare biomimetic polymers to stabilize proteins, specifically, heparin mimics for stabilization of heparin binding proteins

• 3 and 6 million persons are affected by chronic wounds

• Skin ulcers are the most common and pose significant morbidity including risk of infection to patients

• Although over 300 different dressings exist, agents that promote curative wound healing processes are limited (Regranex)

Active agents to heal acute and chronic wounds are needed

Stabilize Active Agent for Wound Healing

Basic Fibroblast Growth Factor (bFGF) and Heparin

• bFGF - crucial role in diverse cellular functions– Embryonic development– Angiogenesis– Tissue and bone regeneration– Development, maintenance of the nervous system– Stem cell self-renewal– Regeneration of aged vocal folds– Wound healing

• Heparin is the natural stabilizer of bFGF– Heterogeneous and difficult to modify

Heparin

bFGF

Faham et al. Science 1996, 271, 1116; DiGabriele,; et al. Nature 1998, 393, 812; Barrientos, et al. Wound Repair Regen. 2008, 16, 585; Whalen, et al. Growth Factors 1989, 1, 157; Nimni, M. E. Biomaterials 1997, 18, 1201. Richard, J. L.; et al. Diabetes Care 1995, 18, 64; Edelman, et al. Biomaterials 1991, 12, 619; Gospodarowicz, et al. J. Cell. Physiol. 1986, 475; Rosenbaum J. Cell Bio Int Rep, 1986, 437; Cariou R. Cell Bio Int, Rep, 1988, 1037; Liekens, et al. Oncol. Res. 1997, 9, 173.

Heparin Mimetic Polymer

Surface plasmon resonance results demonstrate that bFGFbinds to polymer, likely via the heparin binding domain

Growth factor binds to polymer to high salt concentrations like heparin and in cell media

Christman, Vazquez-Dorbatt, Schopf, Kolodziej, Li, Broyer, Chen, Maynard, JACS, 2008, 130, 16585; Kolodziej, Kim, Broyer, Saxer, Decker, Maynard, JACS, 2012, 134, 247

Heparin Mimic =

bFGF Conjugate

Hypothesis: bFGF-heparin-mimicking conjugate will be a more stable than bFGF alone or bFGF-PEGMA

VS. VS.

Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221

Synthesis of Polymers

Mn = 26.1 kDaPDI = 1.11

Mn = 20.9 kDaPDI = 1.28

Heparin mimic

Control

Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221

p(SS-co-PEGMA) bFGF-p(SS-co-PEGMA)

Lane 1: conjugate, reducing Lane 2: conjugateLane 3: bFGF

ESI-GEMMANative PAGE

conjugate

bFGF Conjugate Preparation

Conjugate prepared with one polymer attached to each bFGFNguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221

Conjugate retains full bioactivity

• Assays were done with human dermal fibroblast (HDF) cells• Data were normalized to blank medium at 100%• Experiment was repeated 8 times, including 1 blinded study

100

150

200

250

% C

ell G

row

th

(+)bFGF (-)heparin

(+)bFGF (+)heparin 1μg/ml

(+)bFGF-pPEGMA

(+)bFGF (+)p(SS-co-PEGMA) 1.5 ng/ml

(+)bFGF-p(SS-co-PEGMA)

bFGFbFGF (+) heparin 1 μg/mlbFGF-pPEGMAbFGF (+) p(SS-co-PEGMA) 1.5 ng/mlbFGF-p(SS-co-PEGMA)

bFGF stimulates proliferation of HDF cells in wound healing events

Conjugates have the same initial activity as pristine bFGFNguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221

Conjugate is Stable to Stressors%

Cel

l Gro

wth

100

150

200

250

Storage at 4 °C for 16 hr

100

150

200

250

1

Heat 55 °C for 30 min

(+)bFGF (-)heparin

(+)bFGF (+)heparin 1μg/ml

(+)bFGF-pPEGMA

(+)bFGF (+)p(SS-co-PEGMA) 1.5 ng/ml

(+)bFGF-p(SS-co-PEGMA)

% C

ell G

row

th

100

150

200

2500.1% Trypsin for 16 hr

100

150

200

250

1

pH 4.7 for 16 hr

100

150

200

250

1

1% TFA for 2 hr

Polymer is stable to a wide range of therapeutically relevant stressorsNguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221.

Maynard GroupRational Design to

Enhance Protein andSmall Molecule Drug Delivery

Hydrogels that Stabilize Proteins

Nanoparticles for Protein and Small Molecule Drug Delivery

Polymers that Borrow from Nature to Stabilize Therapeutic Proteins

Polymers that Mimic Nature to Stabilize and Enhance Activity

Nguyen, et al., Nature Chemistry, 2013, 5, 221

Lee, et al., Biomacromolecules, 2013, 14, 2561Lee, et al., Polymer Chem, 2015, 6, 3443

Matsumoto, et al., ACS Nano, 2013, 7, 867

PEG Alternatives for Stabilization

Pan, H. Z.; Sima, M.; Yang, J. Y.; Kopecek, J. Macromol. Biosci. 2013, 13, 155.

Comb and branched PEG-like polymerspolyHPMA

Kochendoerfer et al. Science, 2003, 299, 884

Polyzwitterions

Keefe, A. J.; Jiang, S. Nat. Chem. 2012, 4, 59.

Goals of this research:

Trehalose glycopolymersfor general stabilization as a PEG alternative

Introduction of Trehalose• Trehalose is a natural alpha-linked disaccharide formed by an two α,α-1,1,-

linked glucose units

• Generally regarded as safe (GRAS) and utilized as a food additive –metabolizes to glucose in humans

• Stabilizes and protect biological structures against environmental stresses such as desiccation, freezing, osmotic shock and oxidation

Sakurai, M. Water and Biomolecules: Physical Chemistry of Life Phenomena 2009, 219

http://www.wired.com/2014/03/absurd-creature-week-water-bear/

http://waynesword.palomar.edu/pljuly96.htm

Synthesize Trehalose Polymers - PolyProtek™Hypothesis: Trehalose polymers will be an effective protein stabilizer, both added and as a conjugate

Monomer synthesis is straightforward and polymerizable unit is easily varied, as well as linkage point

Example Polymers:

Lee, Lin, Lau, Hedrick, Bat, Maynard, Biomacromolecules, 2013, 14, 2561

Synthesis of Trehalose Monomer

Baer; Radatus, Carbohydrate Research 1984, 128, 165

1,2

3 45

6 7 8 9 10

11,12,13,14,15,16,17

1819

ka b

c de

f ghi j

lm n o p q

rs

tu

vx

w

y z

x

18

1016

13

8O O

9

O11

15

1214

OHh

OHm17O

7O

19OHl2

54

14

53

6Hd

Hf

Hc

HkHjHo Hp

Hy

OHi Ht

HzOHg

Hq

Hb

Ha

Ha

Hb

HwHe

Hs

Hx

Hr

HuOHn

Hv

13C

1H

Synthesis of Polymer

Different molecular weights are prepared by changing initial conditionsMancini, Lee, Maynard JACS 2012, 134, 8474

Samples: Conjugates and Excipients

Polymers were conjugated to thiolated lysozyme and the conjugates purified by FPLC. The activity of the protein was tested after lyophilization and heat stress. The results were also determined for adding polymer to the protein and compared to trehalose and PEG.

Mancini, Lee, Maynard JACS 2012, 134, 8474

Comparison to Trehalose and PEG

Trehalose polymer, both covalently attached and added as an excepient, is much better at stabilizing proteins to heat and lyophilizationcompared to PEG and trehalose

Mancini, Lee, Maynard JACS 2012, 134, 8474

PolyProtek Mn (by NMR) = 15.4 kDa; PDI = 1.13

Why Are the Polymer Better Than Trehalose?

Do the polymers have trehalose activity for their stabilization?

Do they have nonionic surfactant character?


Hm(J/g)

Hc(J/g) ∆Hm (J/g) ∆Hc (J/g)

Water 303.6 -270.8 - -

1 mol% Trehalose 207.6 -199.5 -96.0 71.3

2 mol% Trehalose 158.3 -154.5 -145.3 116.3

3 mol% Trehalose 100.4 -112.1 -203.2 158.7

1 mol% Trehalosein P3

220.7 -205.4 -82.9 65.4


153.4 -139.5 -150.2 131.3


107.6 -104.5 -196.0 166.3

Differential Scanning Calorimetry Results

Results show that PolyProtek™ has trehalose’s ability to suppress water crystallization similar to trehalose


Circular Dichroism StudiesHRP β-Gal

CD suggests a chaperone-like activity for the polymer with regard to heat; the polymer also prevents aggregation.


Overview of Maynard Current and Future Work

Animal Feed Enzyme Industry is Growing

The global feed enzyme market reached $550 million in 2011 and is projected to grow to $1.2 billion by 2018

The growth has been driven by more efficient enzyme production, making the use of feed enzymes cost effective

Adeola, O., Cowieson, A. J. J. Of Animal Sci., 2011, 89, 3189.Bedford, M. R., Patridge, G. G. Enzymes in Farm Animal Nutrition, 2011, 307.

Phytase is a Major Enzyme in the Feed Industry

Phytase is a hydrolase that converts phytate in plant-based feeds into inorganic phosphorus

Simple-stomached animals such as pigs need phytase for digestion of phytate in their diet

Phytase accounts for 60% of the feed enzyme market

Adeola, O., Cowieson, A. J. J. Of Animal Sci., 2011, 89, 3189.Lei, X. G. et al. Ann. Rev. of Animal Biosci., 2013, 1, 283.

Phytase

H2O+

6 PO4

OPO32-

OPO32-

OPO32-

OPO32-

OPO32-

OPO32-

HO OHOH

HOOH

OH

Phytate Inositol

Challenges and Strategies to Stabilizing Phytase

Pelleting is the most common processing method for animal feeds due to its efficiency

However, phytase is thermally unstable to heating during the pelleting process (71 – 90 °C)

Current strategies involve using protein engineering to enhance thermostability of phytase or coating the phytase with a protectant

These strategies can increase costs

Hypothesis: Trehalose-based hydrogel materials will stabilize phytase and other feed enzymes to high temperatures

Thomas, M., van der Poel, A. F. B. Anim. Feed Sci. Tech., 1996, 61, 89; Nahm, K. H. Crit. Rev. Env. Sci.Tech., 2002, 32, 1; Lei, X. G. et al. Ann. Rev. of Animal Biosci., 2013, 1, 283.

Reaction yielded various regioisomers of mono-, di-, and tri-substituted trehalose.

Synthesis of Trehalose-based Hydrogel

Trehalose Mono-substituted

Di-substitutedTri-substituted

Time (min)

LC-MS1:1 H2O, MeOH

OOH

O

OHO

HO

OHO

HOOH

OH

OHOH

O

OHO

HO

OHO

HOOH

OHCl

+ NaOH

DMSO, 25 oC, 24

h

+

OOH

O

OHO

HO

OO

HOOH

OH

+ regioisomers and higher substituted

products

Lee, Ko, Lin, Wallace, Ruch, Maynard, Polymer Chemistry, 2015, 6, 3443

Gelation of the Substituted Trehalose Mixture

The substituted trehalose mixture was polymerized by redox initiated radical polymerization, and was subsequently grounded and washed

Hydrogel gel preparation is easily, cheap, and scalable

(a) (b)

(a) Hydrogel before wash and (b) after grinding and washing the hydrogel

OOH

O

OHO

HO

OHO

HOOH

OH

+

OOH

O

OHO

HO

OO

HOOH

OH

+ regioisomers and higher substituted

products

APS/TEMED

H2O, 25 oC, 24

h


Hydrogel Properties

Pore sizes large enough for protein to penetrate throughout gel

Protein is readily released

SEMProtein Release

Confocal


Stabilization to Heat

Heat at 90 , 1 min(54 wt% H2O)

Trehalose hydrogel stabilized phytase against heat at all gel-to-phytase weight equivalents tested; the optimal ratio is 10 wt eq to retain activity while minimizing amount of material used

Gel is cheap, easily scalable and highly effective enzyme stabilizer


Conclusions

Rational design of polymers leads to desirable properties:

Heparin mimicking polymer stabilizes bFGF to a wide range of therapeutically relevant stressors and better than the PEGylatedversion.

Trehalose side chain polymers are excellent protein stabilizers, better than PEG or trehalose, and hydrogels are cheap, scalable and able to effectively stabilize animal feed enzymes

Fertile Ground

Polymers are fertile ground with untapped potential in the plant and food industries:

-Polymers to stabilize feed enzymes and peptide antibiotics

-Hydrogels to minimize use of water and maximize delivery of nutrients and fertilizer to plants

-Drug delivery of antifungals, antibacterials, and pesticides to certain locations in a plant or tree and for sustained or timed release

-Polymers to enhance the storage and stability of food

Acknowledgements

Collaborators:• Prof. Joe Loo (UCLA)• Dr. Frank Ruch and Peter Wallace

(Phytex)

Students and Postdocs (current position):• Dr. Erhan Bat (Middle East Tech U)• Cait Decker• Dr. Sung-Hye Kim (Celanese-Ticona)• James Hedrick (Northwestern)• Jeong Ho Ko• Uland Lau• Dr. Juneyoung Lee (Caltech)• Dr. En-Wei Lin (Nalco)• Dr. Yang Liu (Chapman U)• Dr. Rock Mancini (Wash State U)• Dr. Kathy Nguyen (Pearce College)• Dr. Darice Wong (UCLA Bioengineering)

$$: NSF (CHE); NIH (NIBIB); Phytex