Abstract:
A method for lowering the extractable protein content in a natural rubber latex to below 30 parts per million (ppm) comprises the steps of treating by washing a rubber article, such as a rubber glove, with an aqueous solution containing a soluble silicate and, optionally, a protease. The methods claimed effectively reduce the allergenicity of the articles so treated.

Description:
TECHNICAL FIELD 
     This invention relates to the production of natural rubber latex articles having a low protein content. 
     BACKGROUND ART 
     Articles made from natural rubber latex generally contain protein, and this may give rise to a variety of undesirable effects in the finished article, including allergic reactions in articles intended for use in contact with human skin, e.g. surgical gloves, medical catheters, household gloves and condoms. 
     The protein content in rubber latex articles is commonly expressed in two different ways: as total extractable protein (EP) and as protein antigen. The total extractable protein (EP) can be measured, e.g., by the Lowry protein assay. The protein antigen expresses the allergenicity of the protein in the article; it may be measured, e.g., by the ELISA assay method. 
     A number of methods have been proposed to reduce the protein content or to alleviate the allergic reactions in articles made of natural rubber latex. Thus, Novo Nordisk A/S in Research Disclosure, Mar. 10, 1992, No. 335102 discloses that treatment of latex or latex goods with a protease is effective for changing the proteins to non-antigens and also for reducing the amount total protein in latex goods such as gloves. AU-A-44456/93 (Sumitomo Rubber Ind. &amp; Kao Corp.) discloses a treatment of natural rubber latex with a protease, an anionic surfactant and/or a non-ionic surfactant. AU-A-63069/94 and AU-A-63070/94 (both to Sumitomo Rubber Ind. &amp; Kao Corp.) disclose a treatment of a natural rubber latex with a protease and a surfactant, followed by the addition of, an anionic, amphoteric or non-ionic surfactant, oligomer or polymer, to improve the mechanical stability. 
     SUMMARY OF THE INVENTION 
     Surprisingly, we have found that the extractable protein content of a rubber latex article can be reduced by washing the article with a solution containing a soluble silicate. In a highly preferred embodiment, the solution also comprises a protease which serves to further reduce the extractable protein content and particularly to make the remaining protein less antigenic. With a combination of silicate and protease it is possible to reduce the protein antigen content to below the detection limit of the commonly used ELISA method. 
     Accordingly, the invention provides a method for producing a natural rubber latex articles, comprising washing the article with a solution comprising a soluble silicate and optionally a protease. 
     Another aspect of the invention provides a composition comprising a protease and a soluble silicate, in amounts which make the composition effective for use in lowering the extractable protein content of an article made of natural rubber latex. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Natural Rubber Latex Article 
     The process of the invention is applicable to reducing the content of extractable protein in any article made of natural rubber article, particularly latex-dipped products. Some specific examples are surgical gloves, medical catheters, household gloves and condoms. 
     Soluble Silicate 
     The soluble silicate may be a crystalline alkaline silicate or a colloidal neutral silicate. It is preferably an alkali metal silicate, particularly a sodium silicate, preferably having a ratio of Na 2  O/SiO 2  in the range from 2:1 to 1:3.75. Some specific examples are: 
     
         ______________________________________Chemical name  Empirical formula                        Na.sub.2 O/SiO.sub.2  ratio______________________________________Sodium orthosilicate          Na.sub.4 SiO.sub.4                        2:1Sodium sesquisilicate          Na.sub.6 Si.sub.2 O.sub.7                        1.5:1Sodium metasilicate          Na.sub.2 SiO.sub.3                        1:1Colloidal neutral silicate          --            1:1.6 to 1:3.75______________________________________ 
    
     A suitable concentration of the soluble silicate is 0.2-10 g/l, preferably 1-5 g/l, in the solution used in the process of the invention or 10-90% by weight of the composition of the invention. 
     Protease 
     Any type of protease known to be effective on latex extractable protein or effective as a detergent additive can be used in the invention. An alkaline protease is preferred, e.g. an alkaline protease derived from Bacillus or Aspergillus, particularly a subtilisin. Some preferred proteases are Subtilisin 147 and Subtilisin 309 described in WO 89/06279 and Subtilisin Carlsberg. Also, a mixture of two or more proteases can be used, e.g. a mixture of 5 proteolytic components from Aspergillus oryzae described in WO 94/25580. Suitable protease preparations are commercially available from Novo Nordisk A/S under the trade names Alcalase®, Savinase®, Esperase® and Flavourzyme™. They are available both in liquid form and as non-dusting granulates. 
     The required concentration of the protease depends on the washing conditions (time, temperature etc.). For 5-15 minutes washing at 30-50° C., a suitable protease concentration corresponds to 0.5-100 KNPU/L, particularly 10-100 KNPU/L in the solution used in the process of the invention or 0.2-2 KNPU/g of the composition of the invention. KNPU is a protease activity unit, defined in U.S. Pat. No. 3,840,433. 
     Optional Ingredients 
     The removal of extractable protein may be enhanced by further adding a surfactant, preferably an amphoteric surfactant, and/or a second soluble electrolyte (in addition to the soluble silicate). 
     However, the use of an amphoteric surfactant tends to make the rubber latex articles more sticky and to increase foaming of the solution. It may therefore be preferred to avoid the use of a surfactant. 
     Also, other conventional additives may be included to enhance the effect of the protease, such as polyethylene glycol (PEG) and polymers like a polyacrylate or polyvinyl pyrrolidone. 
     Surfactant 
     If it is desired to use a surfactant, this may be amphoteric, anionic, non-ionic or a combination of these. It is particularly preferred to use an amphoteric surfactant or a combination of anionic and non-ionic surfactant. 
     A suitable concentration of the surfactant is 0.2-10 g/l, particularly 1-5 g/l in the solution used in the process of the invention or 3-30% by weight of the composition of the invention. 
     Amphoteric Surfactant 
     The amphoteric surfactant may be a propionate, e.g. tallow ampho-poly-carboxy-propionate (e.g. Ampholak™ 7TY); an imino-dipropionate, e.g. complex coco-imino-dipropionate (e.g. Ampholak YCE); an alkyl betaine, e.g. C-cetyl betaine having the formula (CH 3 ) 3  N +  --CHR--COO (R=cetyl), available under the trade name Aquarex™ NS; an alkylamido betaine; an imidazoline derivative, or an alkyl poly-amino carboxylate (APAC). 
     A preferred amphoteric surfactant is a tallow ampho-poly-carboxy-glycinate, particularly N-tallow alkyl poly-trimethylene poly-carboxymethyl sodium salts, available in aqueous solution as Ampholak™ 7TX from Berol Nobel. It has the formula: 
     
         R--(N--X)--(CH.sub.2).sub.3 --(N--X)--(CH.sub.2).sub.3 --(N--X)--(CH.sub.2).sub.3 --N--X 
    
     wherein R=C 16-18  and X=--CH 2  --COONa. 
     Another preferred amphoteric surfactant is oleo-ampho-poly-carboxy-glycinate, available in powder form as Ampholak X07-SD55 from Berol Nobel. 
     Anionic Surfactant 
     The anionic surfactant may be an α-olefin sulfonate (AOS), E.G. Lion™ AOS, a linear alkylbenzene sulfonate (LAS), e.g. Miranil™, or an alkyl sulphate, e.g. lauryl sulfate or Sipon™ LS from Henkel. 
     Non-ionic Surfactant 
     The non-ionic surfactant may be an alcohol ethoxylate, e.g. Elfapur™ LM 75 S from Akzo Chemie or Dobange™ from Berol Nobel, or it may be a nonyl phenol ethoxylate, e.g. Elmulwin™ W from Bayer. 
     Second Soluble Electrolyte 
     The second soluble electrolyte may be an alkali metal salt (particularly a sodium salt) such as a sulphate, a carbonate, a bicarbonate, a chloride, a phosphate, a citrate or a combination of two or more of these. 
     A suitable concentration for the second soluble electrolyte is 1-10 g/l. 
     Washing Process 
     Suitable washing conditions are 1-60 minutes (particularly 5-30 minutes) washing at 20-70° C. (particularly 30-60° C.) and pH 10-12.5 (particularly 10.5-12). A suitable weight ratio between the natural rubber latex articles and the washing solution is in the range from 1:20 to 1:5. 
     The washing may be performed with gentle mechanical stirring. The washing is typically preceded by a prewashing with water and followed by rinsing with water and drying. As mentioned above, there is generally less foaming if the use of a surfactant is avoided, and this makes the rinsing more effective. 
     Composition for Use in Protein Removal 
     The composition of the invention may be provided in powder, liquid or slurry form. A specific example of the formulation is a powder formulation (to be used at a dosage of about 17 g/l) consisting of Savinase 8.0T (protease granulate with an activity of 8.0 KNPU/g) and sodium metasilicate at a weight ratio of 1:2 to 1:4. 
    
    
     EXAMPLES 
     In the Examples 1-4 and Comparative Example 1, natural rubber latex gloves were treated at the following conditions, unless otherwise noted: 
     Natural Rubber Latex Gloves 
     Weight: 9 g per glove 
     Extractable protein content: 600-1200 ppm (measured by the Lowry method). 
     The gloves used in each example were from the same batch. 
     Pre-wash 
     Temperature: ambient (22° C.) 
     Time: 15 minutes 
     Glove/water ratio: 1:10 w/w (e.g. 50 gloves of 9 g each washed with 4.5 l of solution). 
     Stirring: manual 
     Liquid: Tap water 
     Washing 
     Temperature: 50° C. 
     Time: as noted 
     Glove/water ratio: 1:10 w/w 
     Stirring: manual 
     Liquid: distilled water with additives as given in each example 
     Rinsing 
     Temperature: ambient (22° C.) 
     Time: 5 minutes 
     Liquid: running tap water 
     Stirring: manual 
     Drying 
     Temperature: 70° C. 
     Time: 30 minutes 
     Extractable Protein Determination 
     Determination of extractable protein in ppm by Lowry assay. 
     Ingredients Used 
     The following ingredients were used in the examples. Concentrations are given in w/w %. 
     Protease: Savinase 16.0 L NR, protease activity 16.0 KNPU/g 
     Silicate: Sodium metasilicate 
     Amphoteric surfactant: Ampholak 7 TX 
     Non-ionic surfactant: alcohol ethoxylate (Elfapur or Dobange) 
     Anionic surfactant: alkyl sulfate (Sipon) 
     Sulphate: Sodium sulphate 
     Example 1 
     Natural rubber latex gloves were washed with the solutions shown below. The results are given below as ppm of extractable protein after 5 and 15 minutes. 
     
         ______________________________________Test No.   1       2      3     4    5     6    7______________________________________Silicate   1       1      1     1    1     0.5  0.5Protease   0.1     0.1    0.1   0.1  0.1   0.1  0.1Amphoteric   --       --    0.5   0.5  1     1    0.5Non-ionic   --      --     --    --   --    --   0.5Sulphate   --      0.3    --    0.3  --    0.3  0.35 min.  40      37     23    26   25    27   2415 min. 31      29     22    22   21    24   22______________________________________ 
    
     It is seen that the extractable protein content can be brought down to about 30 ppm after 15 minutes washing with silicate and protease, and that it can be brought further down to about 22 ppm by addition of amphoteric surfactant. 
     Example 2 
     Natural rubber latex gloves were washed with the solution shown below, with or without protease: 
     
         ______________________________________  Silicate         0.3  Protease         0 or 0.1  Anionic         0.5  Nonionic         0.1  Sulphate         0.3______________________________________ 
    
     Total extractable protein was measured by Lowry, and the extractable protein antigen was measured by ELISA. Results: 
     
         ______________________________________  Without protease                With proteaseTime, min.    Lowry, ppm              ELISA, ppm                        Lowry, ppm                                ELISA, ppm______________________________________ 5       38        0.6       33      010       35        0.35      30      015       24        0.65      25      0______________________________________ 
    
     The results show that silicate together with surfactant and sulphate was effective in removing the extractable protein (Lowry), increasing with longer washing time to a level below 30 ppm. The addition of a protease was very effective in removing the allergenicity of the remaining protein to a level below the detection limit, and the protease also accelerated the reduction of extractable protein. 
     Example 3 
     Repeated washing tests were carried out with 3 different formulations, as shown below, using gloves of different batches. The results are given as average extractable protein content after 5, 10 and 15 minutes washing. 
     
         ______________________________________Test No.   1             2      3______________________________________Silicate   0.3           0.3    0.3Protease   0.1           0.1    0.1Anionic    0.5           0.6    0.5Non-ionic  0.1           0.1    0.1Sulphate   0.3           0.3    0.3Repetitions      10            12     45 min.     42            38     3710 min.    36            34     3215 min.    34            29     28______________________________________ 
    
     The results show that the extractable protein content can be reduced to about 30 ppm by 15 minutes washing with silicate and protease together with surfactants and sulphate. 
     Comparative Example 1 
     For comparison, gloves were washed without silicate, in water with or without protease (0 or 0.1% Savinase). The total extractable protein was measured by Lowry analysis, and the extractable protein antigen was measured by ELISA. Results: 
     
         ______________________________________  Without protease                With proteaseTime min Lowry ppm ELISA ppm Lowry ppm                                ELISA ppm______________________________________0        739       41.4      739     41.45        58        3.2       101     &lt;0.615       57        2.2       77      &lt;0.630       43        1.2       80      &lt;0.6______________________________________ 
    
     The results show that a treatment with protease alone is effective in removing protein antigens, but does not reduce the total amount of extractable protein. 
     Example 4 
     Natural rubber latex gloves were washed for 15 minutes with the solutions shown below. In this experiment, no pre-wash was used. The results are given as ppm of extractable protein. The control (untreated gloves) contained 750 ppm of Lowry protein. 
     
         ______________________________________         Invention                Reference______________________________________Silicate        1.0      --Protease        0.1        0.1Amphoteric      0.5      --pH              12       12ppm protein     30       72______________________________________ 
    
     The results show a significant reduction of the protease content by the use of soluble silicate, compared to protease alone. 
     Example 5 
     In this experiment, natural rubber latex gloves were washed in a washing machine, using the following procedure. The glove:water ratio was 1:10, and the time for 1 cycle wash was 1 hour. 
     
         ______________________________________Step   Operation       Time (min.)                            Temperature______________________________________1      Pre-wash        6         302      Drop bath       1         --3      Main wash       15        504      Drop bath + spinning                    1.5     --5      Rinsing         5         306      Drop bath + spinning                    1.5     --7      Powder re-coating +                  5         30  spinning8      Drying          30        70______________________________________ 
    
     With each of the solutions shown below, the pH of the solution and the extractable protein (Lowry) after washing were measured. The control (untreated gloves) contained 750 ppm of Lowry protein. Results: 
     
         ______________________________________                                 LowrySilicate    Amphoteric Protease pH    (ppm)______________________________________Invention  0.5      --         --     10.5    22.5  0.5      0.5        --     10.6    14.5  0.5      --         0.1    10.5  18  0.5      0.5        0.1    10.6  17  1.0      0.5        0.1    11.8    10.5  0.3      --         0.1    10.2  28  0.2      0.2        0.1    10.2  18  0.1       0.05      0.1    10.2  25Reference  --       --         --      9.5  74______________________________________ 
    
     The above results demonstrate the excellent performance of the soluble silicate. The protein content could be brought down to below 30 ppm by the use of 0.5% silicate alone or by a combination of silicate with surfactant and/or protease. 
     The stickiness of the gloves after the washing was compared qualitatively. It was found that those treated with silicate together with amphoteric surfactant were more sticky than those treated with silicate alone, indicating that it may be preferable to avoid the use of a surfactant.