Recrystallized insulin

Stock Solution 1 2.18 g of recrystallized insulin are dissolved in 25 ml of 0.1 N hydrochloric acid, and distilled water to a volume of 125 ml is added. 

Figure 8.3. Chromatographic purification of recrystallized insulin on a Sephadex G-50 gel filtration column. Separation of high molecular mass proteolytic enzymes, as well as proinsulin and some very low molecular mass material, is obvious. Insulin elutes from the column as a single peak, hence the term single peak insulin ...

The appearance of purified (repeatedly recrystallized) insulins and more recently of monocomponent insulins has brought about a diminution in the rates of local reactions, formerly observed at the site of injection in 40%-56% of cases (Paley and Tunbridge 1952), and generalized reactions such as urticaria or anaphylactic shock (Feinglos and Jegasothy 1979 Jorpes 1949 Kreines 1952 Ljung 1952 Mirouze et al. 1973 Sherman 1954 Wolfromm and Nataf 1965 Liebermann et al. 1971 DE Shazo 1978). 

Joslin EP, Gray H, Root HF (1922) Insulin in hospital and home. J Metab Res 1 651 Jorpes JE (1949) Recrystallized insulin for diabetic patients for insulin allergy. Arch Intern Med 83 363... 

Ljung O (1952) Recrystallized insulin in insulin allergy. Acta Med Scand 48 260 MacCuish AC, Jordan J, Campbell CJ, Duncan LJP, Irvine WJ (1975) Cell-mediated immunity in diabetes mellitus. Lymphocyte transformation by insulin and insulin fragments in insulin-treated and newly-diagnosed diabetics. Diabetes 24 36... 

During the first decade of the insulin era, only an acid solution of an impure form of the hormone was available for therapy. When it became possible to crystallize insulin, the purity, and hence the biological potency, of the hormone improved substantially (Table I). These improvements were mainly achieved by introduction of Ztf+-crystallization (Scott, 1934) and recrystallization methods, the latter based on the observation that insulin recrystallized several times was better tolerated by patients suffering from allergic reactions (Jorpes, 1949). Until the late 1960s, recrystallized insulin was considered to be an essentially pure substance, but the introduction of new analytical methods made it possible to detect the presence of significant amounts of protein impurities by disc electrophoresis (Mirsky and... 

By the mid-1930s, commercial insulin was being prepared by crystallization from crude porcine or bovine extracts. The crystallized preparation was generally subjected to a recrystallization step in order to further increase the product s purity. Such preparations are termed conventional insulins (Box 8.2). 

Chromatographic or electrophoretic analysis of conventional insulins generally yields three major fractions or bands a, b and c). Fraction a contains high molecular mass material which can be removed from the product by additional recrystallization steps. The major components of fraction b are proinsulin and insulin dimers, while insulin, as well as slightly modified forms of insulin (e.g. arginine-insulin and desamido-insulin), are found in fraction c. 

Gas [Gas antisolvent] A process for separating dissolved materials by selective precipitation with added supercritical carbon dioxide. First used for recrystallizing the explosive RDX subsequently used for recrystallizing other explosives, pharmaceuticals, fine chemicals, and food products. Another use has been for precipitating insulin particles from dimethylsulfoxide solution. 

Materials. NIPAAm obtained from Fisher Scientific Inc. (Fair Lawn, NJ), was recrystallized from hexane. BMA and AA were purchased from Aldrich Chemical Company (Milwaukee, WI). BMA and AA were purified by vacuum distillation at 57 C / 17 mm Hg and 39 C / 10 mm Hg respectively. 2,2 -azobis-isobutyronitrile (AIBN), purchased from Eastman Kodak Company (Rochester, NY), was recrystallized firom methanol. Bovine insulin, trypsin inhibitor and angiotensin II were purchased fix>m Sigma Chemical Company (St Louis, MO). Human calcitonin was a gift from Suntory Ltd. (Tokyo, Japan). Heavy white mineral oil and decane were purchased fix>m Aldrich Chemical Company (Milwaukee, WI). Acetonitrile, HPLC grade, was purchased from Fisher Scientific Inc. (Fair Lawn, NJ). All other chemicals were reagent grade. 

Acknowledgments This work has been supported by the Fonds de la Recherche Scientifique Medicale, Belgium (Grants n 688 and 840). Toads, Bufo marinus, were generously supplied by Mr. D.R. Fischer, Laboratories Warner, Rio de Janeiro, Brazil, assisted for shipment of the animals by the Ministry of Foreign Affairs of Belgium. Recrystallized ox insulin and excipient were gifts from Novo Laboratories. 

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