Polyvalent immunotherapeutics of high specificty based on modified antibodies and a lyophilized injectable formulation highly safe and effective

Abstract

The invention is related to the production and development of a lyophilized injectable formulation of modified antibodies or their variants, highly specific neutralizers of heterologous mixtures of proteins, peptides and other organic or inorganic components have different specific activities and may include but are not limited to the venoms of venomous animals. For convenience, we refer to venoms, but all type of venoms was included of land and marine animals. It is also addressed to the production method which includes the hyperimmunization of mammals for the production of highly specific antibodies, the modification (fragmentation) and purification process, and finally the injectable formulation conferring thereon properties of high purity and high specificity.

1 . A composition of polyvalent immunotherapeutics of high specificity based on highly specific modified antibodies obtained from mammals with a content of at least 17.7% at approximately 19.7% of specific antibodies, conferring high specificity. 
     
     
         2 . A composition of polyvalent immunotherapeutics of high specificity based on highly specified modified antibodies obtained from mammals requiring at least 6.4 mg to approximately 11.6 mg respectively, necessary to neutralize a milligram of venom utilized in the immunization of mammals. 
     
     
         3 . A composition of polyvalent immunotherapeutics of high specificity based on antibodies modified in accordance with  claims 1  and  2 , wherein the high specificity is characterized because the plasma makes the anti-venom or immunotherapeutics neutralize the same amount of venom with lesser quantities of anti-venom, conferring greater security on the human being. 
     
     
         4 . A method to prepare a composition of highly specific antibodies in mammals, neutralizing complete antigens, heterologous mixtures of proteins, peptides and other organic and inorganic compounds of high quality, comprising:
 a) a dilution of the plasma in three volumes of isotonic saline solution of 0.85%, pre-treated with thimerosal, followed by an adjustment in the pH conditions of approximately 3.5 to 4.0, at a temperature of approximately 18° C. to 20° C.,   b) the addition of an acid solution of pepsin pre-treated and pre-activated to achieve a final concentration of approximately 0.9% at approximately 1.1% of pepsin,   c) to maintain the conditions during approximately 180 minutes in order to complete elimination of the Fc fraction from the immunoglobulins for the production of F(ab′)2, fragments.   d) the checking by means of a SDS-PAGE analysis with a maximum IgG concentration of approximately 2% and a maximum albumin concentration not more than 0.5%,   e) to obtain an enzymatic digest which hydrolizes proteins such as albumin, fibrinogen, coagulation factors of hyperimmune plasma which convert into small peptides with an approximate molecular weight of 15 KDa,   f) to subject the mixture to an addition of aqueous solution of ammonium sulfate at 50% with a final saturation concentration of approximately 35% at an approximate temperature of 2° C. at 8° C. below the 15 KDa,   g) to subject the mixture to an aqueous solution of ammonium sulfate 50% with a final saturation concentration of approximately 35% at an approximate temperature of 2° C. at 8° C., in order to obtain a precipitation of components below 15 KDa.   h) to subject the mixture to storage at 2° C. to 8° C., an pH of 6.8 to 7.0, followed by a diafiltration period in filtering tangential fluid using a polyethersulfone membrane of 50 Kda and a borates buffer, maintaining a diafiltration sheer below 1000 sec−1, in order to thus recover in sterile and pyrogen-free containers, nano-filtering through a filter with a pore size of 20 nanometers, adding cryopreserving and regulating agents of isotonicity such as mannitol 9 mg/mL, Alanine 18 mg/mL and Polysorbate 80 0.1 mg/mL.   i) then the mixture is dosed under aseptic conditions, it is lyophilized at an approximate temperature up to −40° C. in approximately 8 hours, for a primary drying at approximately −5° C. with a speed gradient of approximately +5.2° C./hour, secondary drying at approximately 45° C. in approximately 10 hours at an approximately velocity of 9° C./hour, eliminating approximately 98% of the water content.   
     
     
         5 . The method of  claim 4 , wherein step (c), an efficiency is presented of not less than 95% of F(ab′)2 fragment formation. 
     
     
         6 . The method of  claim 4 , wherein step (h), a balance of the isotonicity of the F(ab′)2 fragments takes place with a isotonic saline solution at 0.85%. 
     
     
         7 . The method of  claim 4 , wherein the soluble and/or insoluble oligomers, dimers, F(ab′) 2 , Fab and components of low molecular weight have a purity greater than 85% of F(ab′)2 fragments. 
     
     
         8 . An lyophilized injectable formulation of  claims 1 ,  2  and  4 , wherein the modified antibodies or their variants, highly specific neutralizers of heterologous mixtures of proteins, peptides and other organic and non-organic components having different specific activities and can include but are not limited to venoms of snakes, scorpions, spiders, mollusks, microorganisms, fish, frogs, insects anemones, corals, or a combination thereof, arising therefrom or produced thereby. 
     
     
         9 . Use of the composition of polyvalent immunotherapeutics of high specificity obtained from mammals of  claims 1  to  3 , in order to prepare a medicament to treat pathologies caused by the biological effect of a venom, toxin or their components of a venomous animal selected from snakes, scorpions, spiders, mollusks microorganisms, fish, frogs, insects anemones, corals, or a combination thereof.