Heather D. Maynard
Department of Chemistry and Biochemistry & California NanoSystems Institute
University of California, L...
Proteins are Important Therapeutic Agents
http://www.drugs.com/stats/top100/2013/sales
Alconcel, et al. Polym. Chem. 2011,...
Proteins Have Formulation Challenges
Proteins are generally unstable and challenging to transport and store
At a minimum, ...
Proteins Have Pharmacokinetic Challenges
Most proteins are injected and rapidly degrade in the body by
natural mechanisms
...
Protein-Polymer Conjugate Therapeutics
• PEGylated proteins – the gold standard
in protein-polymer conjugates
• 10 FDA-app...
Maynard Group
Rational Design to
Enhance Protein and
Small Molecule
Drug Delivery
Hydrogels that Stabilize Proteins
Nanopa...
Biomimetic polymers
Mowery, P.; et al. Synthetic glycoprotein mimics inhibit L-
selectin-mediated rolling and promote L-se...
• 3 and 6 million persons are affected
by chronic wounds
• Skin ulcers are the most common
and pose significant morbidity
...
Basic Fibroblast Growth Factor (bFGF) and Heparin
• bFGF - crucial role in diverse cellular functions
– Embryonic developm...
Heparin Mimetic Polymer
Surface plasmon resonance
results demonstrate that bFGF
binds to polymer, likely via the
heparin b...
bFGF Conjugate
Hypothesis: bFGF-heparin-mimicking conjugate
will be a more stable than bFGF alone or bFGF-
PEGMA
VS. VS.
N...
Synthesis of Polymers
Mn = 26.1 kDa
PDI = 1.11
Mn = 20.9 kDa
PDI = 1.28
Heparin mimic
Control
Nguyen, Kim, Wong, Decker, L...
p(SS-co-PEGMA) bFGF-p(SS-co-PEGMA)
Lane 1: conjugate, reducing
Lane 2: conjugate
Lane 3: bFGF
ESI-GEMMANative PAGE
conjuga...
Conjugate retains full bioactivity
• Assays were done with human dermal fibroblast (HDF) cells
• Data were normalized to b...
Conjugate is Stable to Stressors%CellGrowth
100
150
200
250
Storage at 4 °C for 16 hr
100
150
200
250
1
Heat 55 °C for 30 ...
Maynard Group
Rational Design to
Enhance Protein and
Small Molecule
Drug Delivery
Hydrogels that Stabilize Proteins
Nanopa...
PEG Alternatives for Stabilization
Pan, H. Z.; Sima, M.; Yang, J. Y.;
Kopecek, J. Macromol. Biosci. 2013,
13, 155.
Comb an...
Introduction of Trehalose
• Trehalose is a natural alpha-linked disaccharide formed by an two α,α-1,1,-
linked glucose uni...
Synthesize Trehalose Polymers - PolyProtek™
Hypothesis: Trehalose polymers will be an effective protein stabilizer,
both a...
Synthesis of Trehalose Monomer
Baer; Radatus, Carbohydrate Research 1984, 128, 165
1,2
3 4
5
6 7 8 9 10
11,12,13,14,
15,16...
Synthesis of Polymer
Different molecular weights are prepared by changing initial conditions
Mancini, Lee, Maynard JACS 20...
Samples: Conjugates and Excipients
Polymers were conjugated to thiolated lysozyme and the conjugates
purified by FPLC. The...
Comparison to Trehalose and PEG
Trehalose polymer, both covalently attached and added as an excepient,
is much better at s...
Why Are the Polymer Better Than Trehalose?
Do the polymers have trehalose activity for their stabilization?
Do they have n...
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...
Circular Dichroism Studies
HRP β-Gal
CD suggests a chaperone-like
activity for the polymer with
regard to heat; the polyme...
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...
Phytase is a Major Enzyme in the Feed Industry
Phytase is a hydrolase that converts phytate in plant-based feeds into
inor...
Challenges and Strategies to Stabilizing Phytase
Pelleting is the most common processing method for animal feeds due
to it...
Reaction yielded various regioisomers of mono-, di-, and tri-
substituted trehalose.
Synthesis of Trehalose-based Hydrogel...
Gelation of the Substituted Trehalose Mixture
The substituted trehalose mixture was polymerized by redox
initiated radical...
Hydrogel Properties
Pore sizes large enough for protein to penetrate throughout gel
Protein is readily released
SEM
Protei...
Stabilization to Heat
Heat at 90 , 1 min
(54 wt% H2O)
Trehalose hydrogel stabilized phytase against heat at all gel-to-phy...
Conclusions
Rational design of polymers leads to desirable properties:
Heparin mimicking polymer stabilizes bFGF to a wide...
Fertile Ground
Polymers are fertile ground with untapped potential in
the plant and food industries:
-Polymers to stabiliz...
Acknowledgements
Collaborators:
• Prof. Joe Loo (UCLA)
• Dr. Frank Ruch and Peter Wallace
(Phytex)
Students and Postdocs (...
of 37

Polymers and Hydrogels for Stabilization and Delivery of Animal Feed Enzymes and Other Biologics: Heather Maynard

Professor Heather Maynard, Associate Director of Technology and Development for the California NanoSystems Institute at UCLA, presents "Polymers and Hydrogels for Stabilization and Delivery of Animal Feed Enzymes and Other Biologics" at the Creator Space™ Science Symposium in Chicago.
Published on: Mar 4, 2016
Published in: Science      
Source: www.slideshare.net


Transcripts - Polymers and Hydrogels for Stabilization and Delivery of Animal Feed Enzymes and Other Biologics: Heather Maynard

  • 1. Heather D. Maynard Department 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
  • 2. Proteins are Important Therapeutic Agents http://www.drugs.com/stats/top100/2013/sales Alconcel, 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, sepsis New functions or activities – leukemia, dystonia, thrombosis Targeting/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
  • 3. 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
  • 4. 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
  • 5. Protein-Polymer Conjugate Therapeutics • PEGylated proteins – the gold standard in protein-polymer conjugates • 10 FDA-approved protein-polymer conjugate drugs – exclusively PEGylated proteins • 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-PEG Stimulates production of white blood cells Used in conjunction with chemotherapy
  • 6. Maynard Group Rational Design to Enhance Protein and Small 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, 2561 Lee, et al., Polymer Chem, 2015, 6, 3443 Matsumoto, et al., ACS Nano, 2013, 7, 867
  • 7. 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
  • 8. • 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
  • 9. 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.
  • 10. Heparin Mimetic Polymer Surface plasmon resonance results demonstrate that bFGF binds 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 =
  • 11. 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
  • 12. Synthesis of Polymers Mn = 26.1 kDa PDI = 1.11 Mn = 20.9 kDa PDI = 1.28 Heparin mimic Control Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221
  • 13. p(SS-co-PEGMA) bFGF-p(SS-co-PEGMA) Lane 1: conjugate, reducing Lane 2: conjugate Lane 3: bFGF ESI-GEMMANative PAGE conjugate bFGF Conjugate Preparation Conjugate prepared with one polymer attached to each bFGF Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221
  • 14. 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 %CellGrowth (+)bFGF (-)heparin (+)bFGF (+)heparin 1μg/ml (+)bFGF-pPEGMA (+)bFGF (+)p(SS-co-PEGMA) 1.5 ng/ml (+)bFGF-p(SS-co-PEGMA) bFGF bFGF (+) heparin 1 μg/ml bFGF-pPEGMA bFGF (+) p(SS-co-PEGMA) 1.5 ng/ml bFGF-p(SS-co-PEGMA) bFGF stimulates proliferation of HDF cells in wound healing events Conjugates have the same initial activity as pristine bFGF Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221
  • 15. Conjugate is Stable to Stressors%CellGrowth 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) %CellGrowth 100 150 200 250 0.1% 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 stressors Nguyen, Kim, Wong, Decker, Loo, Maynard, Nature Chemistry, 2013, 5, 221.
  • 16. Maynard Group Rational Design to Enhance Protein and Small 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, 2561 Lee, et al., Polymer Chem, 2015, 6, 3443 Matsumoto, et al., ACS Nano, 2013, 7, 867
  • 17. 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 glycopolymers for general stabilization as a PEG alternative
  • 18. 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
  • 19. 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
  • 20. Synthesis of Trehalose Monomer Baer; Radatus, Carbohydrate Research 1984, 128, 165 1,2 3 4 5 6 7 8 9 10 11,12,13,14, 15,16,17 18 19 ka b c d e f g hi j lm n o p q r s tu v x w y z x 18 10 16 13 8 O O 9 O 11 15 12 14 OHh OHm17 O 7 O 19 OHl2 5 4 1 4 5 3 6 Hd Hf Hc HkHj Ho Hp Hy OHi Ht Hz OHg Hq Hb Ha Ha Hb HwHe Hs Hx Hr Hu OHn Hv 13C 1H
  • 21. Synthesis of Polymer Different molecular weights are prepared by changing initial conditions Mancini, Lee, Maynard JACS 2012, 134, 8474
  • 22. 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
  • 23. Comparison to Trehalose and PEG Trehalose polymer, both covalently attached and added as an excepient, is much better at stabilizing proteins to heat and lyophilization compared to PEG and trehalose Mancini, Lee, Maynard JACS 2012, 134, 8474 PolyProtek Mn (by NMR) = 15.4 kDa; PDI = 1.13
  • 24. Why Are the Polymer Better Than Trehalose? Do the polymers have trehalose activity for their stabilization? Do they have nonionic surfactant character? Lee, Lin, Lau, Hedrick, Bat, Maynard, Biomacromolecules, 2013, 14, 2561
  • 25. 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% Trehalose in P3 220.7 -205.4 -82.9 65.4 2 mol% Trehalose in P3 153.4 -139.5 -150.2 131.3 3 mol% Trehalose in P3 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 Lee, Lin, Lau, Hedrick, Bat, Maynard, Biomacromolecules, 2013, 14, 2561
  • 26. Circular Dichroism Studies HRP β-Gal CD suggests a chaperone-like activity for the polymer with regard to heat; the polymer also prevents aggregation. Lee, Lin, Lau, Hedrick, Bat, Maynard, Biomacromolecules, 2013, 14, 2561
  • 27. Overview of Maynard Current and Future Work
  • 28. 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.
  • 29. 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 OPO3 2 - OPO3 2 - OPO3 2 - OPO3 2 - OPO3 2 - OPO3 2 - HO OH OH HO OH OH Phytate Inositol
  • 30. 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.
  • 31. Reaction yielded various regioisomers of mono-, di-, and tri- substituted trehalose. Synthesis of Trehalose-based Hydrogel Trehalose Mono-substituted Di-substituted Tri-substituted Time (min) LC-MS 1:1 H2O, MeOH OOH O O HO HO O HO HO OH OH OHOH O O HO HO O HO HO OH OHCl + NaOH DMSO, 25 o C, 24 h + OOH O O HO HO O O HO OH OH + regioisomers and higher substituted products Lee, Ko, Lin, Wallace, Ruch, Maynard, Polymer Chemistry, 2015, 6, 3443
  • 32. 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 O HO HO O HO HO OH OH + OOH O O HO HO O O HO OH OH + regioisomers and higher substituted products APS/TEMED H2O, 25 o C, 24 h Lee, Ko, Lin, Wallace, Ruch, Maynard, Polymer Chemistry, 2015, 6, 3443
  • 33. Hydrogel Properties Pore sizes large enough for protein to penetrate throughout gel Protein is readily released SEM Protein Release Confocal Lee, Ko, Lin, Wallace, Ruch, Maynard, Polymer Chemistry, 2015, 6, 3443
  • 34. 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 Lee, Ko, Lin, Wallace, Ruch, Maynard, Polymer Chemistry, 2015, 6, 3443
  • 35. 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 PEGylated version. 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
  • 36. 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
  • 37. 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

Related Documents