COMMERCIALIZATION OF YULEX NATURAL RUBBER LATEX™: THE IMPROVING MARGIN STRUCTURE OF GUAYULE LATEX PRODUCTS

Presented by: Katrina Cornish, Ph.D. Senior Vice President, Research & Development Yulex Corporation 1945 Camino Vida Roble, Suite C, Carlsbad CA 92008 Phone: 760-476-0320 x101 ● Fax: 760-476-0321 ● Email: [email protected]

Dr. Katrina Cornish is the leading U.S. scientific expert on domestic latex production. As Senior Vice President, Research & Development at Yulex, Dr. Cornish oversees the company's ongoing research, development, and validation programs for the commercialization of Guayule latex for hypoallergenic medical devices and specialty consumer products.

Prior to joining Yulex in 2004, Dr. Cornish was the USDA Agriculture Research Service, Western Regional Research Center's highest ranking female scientist and had led the agency's program to develop domestic natural rubber production for 15 years. Her broad-biobased research program at the USDA encompassed rubber biochemistry, molecular biology, immunochemistry, chemistry, polymer chemistry, processing, and bio-based product development aimed towards the biotechnological development of plant species suitable for commercially-viable cultivation for rubber production in the temperate climate of the United States. Dr. Cornish is the sole inventor of process and product patents to produce hypoallergenic natural rubber latex and products from guayule. She began working closely with Yulex in 1997 when the company first licensed her discoveries from the USDA. She has 100 publications and patents of which 70 are related to rubber biosynthesis and production.

In 2004, Cornish received the Good Housekeeping Award for Women in Government in recognition of her inspired leadership and proving that good government can change our lives. In 2002, she was elected a Fellow of the American Association for the Advancement of Science and won a presidential award from the American Chemical Society. In 1998, she was honored with the USDA's Scientist of the Year Award for Outstanding Senior Research Scientist. In 1997, she was recognized by the Agricultural University of Antonio Narro, Mexica for guayule research.

Dr. Katrina Cornish studied at the University of Birmingham, England, where she gained a First Class Honours B. Sc. Degree in Biological Sciences (1978), the John Humpreys Memorial Prize for Plant Biology, and a Ph.D. in Plant Biology (1982) on adaptive mechanisms of salt tolerance in pasture grasses. She then moved to the United States and held post-doctoral appointments in various research fields including the physiology and biochemistry of abscisic acid metabolism and movement in relation to drought stress (Michigan State University - DOE Plant Research Laboratory)., the biochemical and electrophysiological characterization of novel amino acid:H+ contransport systems in developing pea and soybeans cotyledons (Cornell University), photosynthesis in cotton and sugar cane in relation to crop evolution (University of California, Santa Cruz, and University of Arizona), and natural rubber biosynthesis in guayule (Arizona State University). She serves on the editorial boards of Phytochemistry, Industrial Crops and Products and Industrial Biotechnology. She is a Fellow of the American Association for the Advancement of Science, and a member of the American Society of Plant Biologists, the Association for the Advancement of Industrial Crops, the American Chemical Society, the BioEnvironmental Polymer Society and the European Federation of Biotechnologists.

Co-Author: Jali Williams Yulex Corporation

Originally from Los Angeles CA, Jali graduated from California Polytechnic State University, San Luis Obispo with a B.S. in Chemistry, Polymers and Coating Concentration. His work experiences include environmental contamination analysis, failure detection and analysis by SEM-EDX. More recently, he was a Researcher with a major glove manufacturer leading synthetic exam and industrial glove process/formulation development and technology transfer/scale-up exercises. In addition, Jali spent 4 years as a Manufacturing-Based Technology Transfer Liaison in Thailand coordinating transfer and scale-up efforts between Manufacturing, Sales & Marketing, and Research & Development. He currently leads the process and product development effort at Yulex Corporation's Arizona-based manufacturing facility.

Co-Author: Jeffrey A. Martin, President Yulex Corporation

Commercialization of Yulex Natural Rubber Latex™: the improving margin structure of guayule latex products

Katrina Cornish, Jali Williams and Jeffrey A. Martin Yulex Corporation 1945 Camino Vida Roble, Suite C Carlsbad, CA 92008, U.S.A.

Abstract:

Yulex Corporation is currently commercializing the production of “yulex™” a high performance natural rubber latex for use in medical products that are non-allergenic to Type I Hevea latex protein-sensitized individuals. In these markets, tropical latex products pose significant risk to Type I latex allergic individuals, and synthetic polymers have not met the challenge of providing comparable physical performance to natural rubber coupled with reasonable cost. Yulex has built and is operating an automated, continuous-flow, medium-scale pilot processing plant and has its own seed and production crops. As the crop acreage continues expansion, and additional commercial processing plants come on line to meet the latex demand, the profitable use of the resin coproduct and the bagasse byproduct will ensure complete consumption of the crop and provide continuous improvement in margin structure enabling competition in wider market segments.

Introduction:

Natural rubber (cis-1,4-polyisoprene [9003-31-0]) is an irreplaceable, strategic raw material used in enormous quantities by industry, transportation, medicine and defense1-4. The United States is the world’s second largest consumer of natural rubber, which it imports on a scale second only to its petroleum imports. Unlike fossil fuel, the US has no domestic supply of natural rubber, and no longer has a stockpile. The high performance properties of natural rubber cannot be matched by synthetic polymers, and so cannot be replaced by synthetics in many applications. In addition, in 2002, the three largest rubber-producing countries (Thailand, Malaysia and Indonesia, 70% of the global supply) formed a cartel to control production and prices4. Recently, two other rubber-producing countries (India and Vietnam) requested membership. Quite apart from the homeland security aspects of the natural rubber supply, the lack of genetic diversity and disease resistance puts the natural rubber industry at risk - natural rubber is currently produced largely from clonal plantations of Hevea brasiliensis in tropical South-east Asia, and the clones share a closely-related common ancestry. Leaf blight (Microcyclus ulei), a fungal disease, is the principle cause of the failure of the natural rubber industry in South America, and with modern, rapid transportation patterns, could be accidentally introduced into South-east Asia5. In addition, Hevea tropical latex products have given rise to the world-wide occurrence of life-threatening, IgE-mediated, latex allergy caused by the proteins in Hevea latex. Complete protein removal cannot be easily or cheaply achieved, or proven, and, when attempted, negatively impacts latex product performance2,6,7. Thus, additional rubber- producing crops are greatly desired to increase biodiversity, protect supplies, and provide safe alternative rubber products for individuals suffering from Type I latex allergy.

Guayule as an Alternative Rubber Crop

At least 2,500 plant species are known to produce natural rubber but few do this in commercially-viable quantities or polymer molecular weights8,9. In addition, few rubber plants have ever been grown on a large-scale or as crops. A rare exception to this is guayule (Parthenium argentatum Gray), a rubber-producing woody shrub, native to the Chihuahuan desert of Texas and north central Mexico10-16. Guayule should not be confused with “Parthenium weed”, or “Woody weed” (Parthenium hysterophorus L), a plant notorious for its invasive habit and for its production of an allergenic sesquiterpene lactone. Guayule has shown no signs of invasive behavior, despite field trials in many countries over several decades, and fortuitously does not produce the allergenic sesquiterpene lactone17. Guayule rubber was produced commercially in the early part of the 20th century and was a major focus of the Emergency Rubber Project of World War II18,19. These efforts established that guayule produces high molecular weight rubber that can be used to make high quality tires, facts confirmed in the 1980’s when guayule was cultivated and processed in response to the oil crisis and rising synthetic rubber production costs. Unfortunately, by the time the solvent extraction process, employed to extract guayule rubber as bulk rubber, was fully established, the oil crisis was over and guayule rubber was left to compete with the cheapest end of the tropical rubber market. Guayule rubber was much more expensive (ca 5x) and the commercialization effort came to an abrupt end20. The strategic necessity for natural rubber is not, of itself, sufficient impetus to produce rubber from guayule or other alternative rubber-producing crops. A new opportunity for guayule arose in 1991 when the Food and Drug Administration issued a medical alert warning of Type I latex allergies to proteins in natural rubber products21-26. The allergy arose because the enormously increased demand for latex gloves, caused by the institution of Universal Precautions in response to the AIDS epidemic, led to short-cuts in the glove manufacturing process. The new products were made without washing the soluble latex proteins from the gloves – leaving them to be leached out by patients during glove contact with body fluids and mucosal membranes. The problem was compounded by the prevalence of single-use powdered latex examination gloves in hospital settings. Proteins from the gloves migrated post- manufacture to coat the corn starch powder donning agent. Rapid removal of gloves before the powder was dampened by perspiration led to the release of air-borne latex allergens – and hospital workers then constantly breathed them in inducing the high prevalence of latex allergies in this population. At least 400 medical and dental products are made with natural rubber latex and synthetic products lack the desired, and in some cases required, performance properties. Guayule had not been investigated as a commercial source of latex because it does not make its rubber in the form of a tapable latex. Thus, if a cut in made into the stem of a guayule shrub no milky, rubber-containing emulsion bleeds from the incision. However, on a microscopic level, guayule, like latex-producing species, makes its rubber in small rubber particles floating in the aqueous cytosol. However, in guayule these are produced in individual bark parenchyma cells instead of in laticifers (networks of latex-containing living pipes made from anastomized cell systems). Extracting the guayule rubber particles, while maintaining them in the aqueous suspension, generates an artificially-produced natural rubber latex suitable for the manufacture of latex products.

RESULTS

Guayule Latex Proteins and Immunogenicity

Guayule latex contains very little protein (< 2%) compared with tropical latex and far fewer different proteins2,6,27. Mouse and rabbit trials and human clinical trials, including ELISA (enzyme-liked immunosorbent assay), 1-D and 2-D immunoblots, skin-prick tests, RAST (radioallergosorbent) assays, and CAP assays of allergenic protein levels, have demonstrated that guayule latex proteins do not cross-react with anti-Hevea latex protein antibodies at concentrations at least 1000x the amount of protein sufficient to cause a response to Hevea proteins4,6,27-32. Reciprocal tests using animal antibodies (mice and rabbits) also demonstrated that antibodies deliberately raised against extracted and concentrated guayule latex proteins do not cross-react with Hevea latex proteins29,32. The method by which guayule latex is produced ensures that soluble proteins are washed from the latex during the purification process to undetectable levels. The remaining protein is hydrophobic and associated with the rubber particle membranes.

Table 1. Protein content of guayule latex compared with two samples of Hevea latex (three replicates). The total protein in the latices was quantified using the Modified Lowry described in ASTM D5712-05.

Sample Protein (µg/g dry rubber) Hevea, sample 1 9,636 Hevea, sample 2 9,196 Yulex , drum 1 106

Protein assays by Wenshuang Xie and Colleen M. McMahan, USDA-ARS, Albany, CA.

The low protein content of guayule latex and latex products is substantially lower than that of well-leached Hevea latex products, which were used safely for many decades. The low protein levels, coupled with the hydrophobic nature of the proteins, makes it unlikely that large- scale use of guayule latex medical products will cause the wide-spread development of guayule latex allergies. Nonetheless, Yulex is maintaining an active research program in this area, and has recruited a Scientific Advisory Board with a focus on latex allergies, both Type I and Type IV, to ensure that products made from yulex™ remain as safe as possible.

Guayule Latex Production

To produce guayule latex, the rubber particles must be removed intact from the parenchyma cells while maintaining an aqueous suspension14,16,33-35. Thus, the shrub must remain in a hydrated condition through harvest, shipping and storage until homogenized in an alkaline aqueous extraction medium6,36. The rubber particles, which have a specific gravity of slightly less than 1, can then be purified from the homogenate using a series of centrifugation steps and/or flotation with creaming agents34. This process results in an artificially-produced natural rubber latex from which the cytoplasmic and soluble components have been removed. Purified latex appears to remain stable for months37 . The rubber polymers are of high molecular weight2,6,7, the latex is more viscous than Hevea latex at any comparable %DRC38, and it can be used to manufacture high performance products6,39. The Yulex pilot plant is now operating in full production mode and is fed by commercially-grown guayule crops.

Physical Properties

Tensile Properties

Guayule latex requires different compounding chemistry to Hevea latex largely because of the very low protein content. However, rubber chemistry can be exploited to generate glove films of excellent tensile and elongation properties. Samples were dipped on glass formers as follows:

1. A proprietary compound was matured for 3 hours before dipping 2. Formers were preheated to 75 °C 3. Formers were dipped in coagulant at 45 °C with no dwell time. Coagulant consisted of 17% CaNO3, 4% CaCO3, 0.2% surfactants. 4. Coagulant was dried for 1 min at 75 °C 5. The formers were dipped in the compounded latex (33% TSC, room temp.) with 10 count dwell time. 6. The film-coated formers were dried for 6 min at 75 °C 7. Rolled bead 8. The films were leached for 2 min at 50 °C 9. The films were cured for 15 min at 110 °C 10. The films were removed from the formers and chlorinated.

Tensile samples were cut 10 mm wide, perpendicular to former length.

The physical properties of unaged guayule latex glove films compared very well to standard latex gloves (Figures 1 and 2) and are significantly better than synthetic materials as has been reported previously2,6. As a reference, unchlorinated, lightly powdered Hevea latex gloves have a tensile strength of 22-30 MPa, and an elongation to break of 700-800%, whereas nitrile gloves have a tensile strength of 25-35 MPa and an elongation to break of 550-675%. Thus, this low protein, hypoallergenic guayule latex material outperforms the synthetic nitrile material, and has physical properties at least as attractive as Hevea latex. This raises the possibility of yulex becoming a new medical grade of natural rubber latex with the high tensile properties of Hevea latex but without the allergenic proteins in that material.

Figure 1. The effect of sulfur content on the tensile strength of guayule latex films. The solid line represents the median tensiles of eight replicates, and the upper and lower dashed lines the maximum and minimum values obtained. The Hevea (NRL) sample glove films (vertical line) nominally contain 0.75 phr S[JW1].

Figure 2. The effect of sulfur content on the tensile strength of guayule latex films. The solid line represents the median tensiles of eight replicates, and the upper and lower dashed lines the maximum and minimum values obtained. The Hevea (NRL) sample glove films (vertical line) nominally contain 0.75 phr S[JW2].

Swell Test

Table 2. Linear swell tests demonstrate that unaged guayule latex films reach a full state of cure.

Testing Guayule films Guide latex % Linear phr S % Linear Swell Swell Unvulcanized ≥ 160% 0.5 100 Lightly Vulcanized 100% - 159% 1.0 92 Moderately Vulcanized 80% - 99% 2.0 82 Fully Vulcanized ≤ 79% 3.0 80

These results demonstrate that a full state of cure can be obtained using rubber chemistry tailored to the guayule latex. The data indicate that the cross-link density in the 3.0 phr S is greatest as expected with so much sulfur available.

Modulus

The modulus reaches a maximum at 2 phr sulfur indicating an optimal ratio of the compound to the sulfur. These films have almost as high a cross-link density as the 3 phr films (Table 2). The maximum modulus at 2 phr indicates a maximization of mono-sulfidic links compared with that of the 3.0 phr films, where the additional sulfur allows more poly-sulfidic links which are softer in a macroscopic sense leading to the lower modulus observed.

Figure 3. The effect of sulfur content on the modulus of guayule latex films. The solid line represents the median tensiles of eight replicates, and the upper and lower dashed lines the maximum and minimum values obtained. Latex Rheology

It has previously been observed38 that guayule latex is more viscous than Hevea latex at any particular %DRC, when viscosity was determined using plate-to-plate rheometry.

Yulex 43% Hevea 43% Hevea 61%

Figure 4. Viscosity was determined using a Brookfield LVDV-II+ Viscometer

This property also was observed using a Brookfield LVDV-II+ viscometer in which viscosity is measured by the resistance to a rotating spindle. The higher viscosity may be attributed to the larger particle size of the latex6,27,38, but whatever its root cause it has implications in dipping processes – a higher viscosity may lead to better pick-up, lower residence time, faster line speeds, etc.

Commercialization

The Primary Product

The exclusive licenses to the USDA patents on the latex production process14 and the guayule latex itself16 were awarded to Yulex Corporation in 1997. Currently, Yulex has its own seed and production crops and an automated pilot processing plant. In June, 2005, Yulex entered into a five-year distribution agreement with Centrotrade and has begun latex shipments and commercial sales. The value of the latex in high-end medical uses, such as catheters, surgical tubing, condoms and surgical gloves, is sufficient that guayule production can be supported by the latex stream alone. Petroleum-based materials will continue to have their profit margins squeezed, while the future of guayule latex improves with the inevitable economies of scale and the profitable use of the remaining biomass.

The first 55 gallon drum of yulex™, June 2005.

Table 3. Yulex Product Specification compared with two types of Hevea latex.

Hevea Hevea Yulex First Drum ASTM Spec D1076, Type 1 ASTM Spec D1076, Type 2 Centrifuged NRL Creamed NRL 050525-D01 Total Solids Content (%) 61.3% min 66.0% min 45.1 Dry Rubber Content (%) 59.8% min 64.0% min 42.8 Total Alkalinity, KOH as % Latex 0.6% min as NH3 0.55% min as NH3 0.13 Viscosity @ 43% TSC, cps No requirement No requirement 148 Sludge, weight % 0.10 % max 0.10 % max 0.003 Coagulum, weight % 0.05% max 0.05% max 0.003 KOH number 0.80 max 0.80 max 0.09 pH No requirement No requirement 10.9 Mechanical Stability 650 min @ 55% TSC 650 min @ 55% TSC Pending Copper (ppm) 8 ppm max (dw rubber) 8 ppm max (dw rubber) 2.7 Manganese (ppm) 8 ppm max (dw rubber) 8 ppm max (dw rubber) 0.24 Magnesium (ppm) No requirement No requirement 16.9 Density (Mg/m3) No requirement No requirement 0.95 Color No pronounced blue or grey No pronounced blue or grey Off-white Odor No putrifactive odor No putrifactive odor Ammonia

Tests were performed in accordance with the procedures described in ASTM D1076, where applicable. Items in grey are speculative or as yet there are no data in hand based on the current process. ASTM specification values are relative to type 1 centrifuged and type 2 creamed NRL. Also, we expect that plant breeding40, genetic engineering41 and improved agronomics42 will enhance latex yields and profitability. It seems reasonable to project that guayule latex will eventually be able to compete with Hevea in the bulk rubber markets – it has been proven that guayule rubber bulk properties and acceptable for the high performance market, for example43.

The Coproduct

Our goal, which we consider a necessary one, is to produce latex from sustainable, renewable, environmentally benign guayule production and processing. This includes a requirement to utilize the entire guayule crop, not just the latex component. The guayule shrub produces as much, if not more, acetone-soluble resin as it does latex. Many uses have been identified for the resin, including in bio-based solventless adhesives, biopesticides, antifungal agents, termitic antifeedants, pharmaceuticals, coatings, and paper, as have recently been reviewed44. The profitable utilization of the guayule resin coproduct will spread the cost of guayule production across a wider product base than that enjoyed by the latex alone and will allow latex prices to drop significantly permitting entry into less premium markets. A most promising avenue for commercialization of the guayule resin fraction lies within its excellent bioadhesive properties. The rubber itself is an important component of adhesives but the resin is an excellent tackifier for the rubber. Guayule resin possesses unique characteristics including the capability to bond under water to a variety of substrates and with a viscosity that is significantly lower than other tackifying resins currently used in both solvent-based and solventless adhesives (research by Dr. Ronald Gumbs, Gumbs Associates, Inc., New Jersey, in collaboration with Yulex Corporation). When resin is added to guayule rubber, both the peel and tack increase with resin concentration. Also, guayule rubber is soluble and doesn’t have to be masticated to reduce the gel content before it can be formulated for use in solvent-based adhesives. Guayule resin-based adhesives, including laminating adhesives and pressure sensitive adhesives, were made with up to 89% w/w% guayule resin. These adhesives exhibited excellent physical properties and a study found that harvest date, and post harvest storage had no effect on peel adhesion. Peel adhesive strengths of 4-6 lbs/linear inch for Mylar to stainless steel were obtained, which are in the same range as those reported by Sartomar, 3M and Eastman Chemical. Guayule adhesives are far superior to most other biobased adhesives, such as fish, animal, casein, soybean, blood and starch adhesives. Although guayule adhesives can bond underwater, they cannot compete with the strength of barnacle glue. However, the ability to bond underwater is a property not shared by 3M products.

The Byproduct

Cost-effective consumption of the lignocellulosic bagasse byproduct into products and fuels will ensure steadily improving guayule economics. Thus, the more the high cost of agriculture is shared by the different product streams the lower the sales price demanded by the latex. It is worth noting that guayule is actually a hard wood, not a soft wood like pine, and so particle boards with enhanced performance properties can be made from the bagasse44. Guayule bagasse appears to have fairly similar amounts of cellulose, hemicellulose and lignin although considerable variation in the different plant components has been apparent among results from different laboratories. We have, nonetheless, noted that the percentage of rubber-containing bark to woody plant core remains relatively constant across the wide range of stem diameters in the guayule shrub.

Table 4. Composition of guayule and of two types of guayule bagasse

Fraction Whole shrub Bagasse after latex Bagasse after rubber extraction and resin extraction Rubber (including 8-10% 0.5% 0% latex) Resin 8-12% 9-14% 0% Cellulose 30% 33% 38% Hemicellulose 20-30% 22-33% 30-38% Lignin 12-30% 32% 20-30% Ash 3% 4% 4%

The rubber and resin have a high fuel value and a range of biofuels can be produced from the deresinated bagasse. We hope to explore these avenues further with suitable industrial partners.

Table 5. Fuel values of guayule materials compared to some standard feedstocks.

Source Btu/lb

Guayule 11,000 Guayule bagasse 7,810 Guayule bagasse + resin 9,720 Guayule resin 16,300 Pinewood and kenaf 8,000 Switchgrass 7,750 Wheat straw 8,060 Biodiesel 18,830 – 19,646 Ethanol 13,307

CONCLUSIONS

Commercial production of low protein, hypoallergenic guayule latex is a reality. Researchers have investigated a plethora of approaches seeking ways to best exploit and consume the crop4,43,44, and research into new applications and uses for the latex, the resin coproduct and the lignocellulosic byproduct is continuing in private, academic and government laboratories. Guayule latex can be used to make high quality medical products and substitute for latex from H. brasiliensis and for the best of the synthetic polymers. However, latex characterization also has revealed novel properties that affect manufacturing processes and product quality. Some of these properties may lead to natural rubber products superior to those currently available. Continuing research to enhance yield, to exploit novel polymeric properties, and on the utilization of the coproducts and bagasse, not only will increase the profitability of guayule production. This approach will allow conversion of the entire guayule crop to a plethora of products, while essentially eliminating the negative environmental impact of agricultural waste. Thus, guayule is positioned to rapidly expand as a model industrial crop feeding a new generation of biorefineries capable of producing a wide range of bioproducts and biofuels.

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