Innovative, highly specific, multi-purpose immunotherapies based on modified antibodies and a highly effective and safe injectable freeze-dried formulation

Abstract

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

Description

INNOVATIVE HIGH-SPECIFICITY MULTI-PURPOSE IMMUNOLOGICAL THERAPIES BASED ON MODIFIED ANTIBODY AND A HIGHLY EFFECTIVE AND SAFE INJECTABLE LYOPHILIZED FORMULATION. FIELD OF THE INVENTION The present invention relates to highly specific, polyvalent immunotherapies, antibody modification, and the production process involved. BACKGROUND OF THE INVENTION Poisoning by venomous animals is recognized as a public health problem in certain regions of the world where human-animal interaction is frequent. We can consider animals that produce pharmacological substances that can interfere with our survival as venoms and / or venomous animals. The most studied venoms are those from snakes, scorpions, spiders, mollusks, and microorganisms. However, other venomous animals exist, such as fish, frogs, insects, anemones, and corals, among others.Antidotes based on manipulating the mammalian immune system have been used to counteract the effects of venom, with immunoglobulins, or antibodies, playing a central role. In 1888, Émilie Roux and Alexandre Yersin demonstrated that the blood of animals immunized against diphtheria toxins provided protection to animals exposed to the toxins. In 1890, Emil von Behring and Shibasaburo Kitasato confirmed the transfer of passive immunity against diphtheria and tetanus toxins; this year is considered the beginning of serotherapy and also marks the first generation of antivenoms, with the second generation consisting of immunoglobulins purified from serum and the following generation composed of immunoglobulin fragments. The production of F(ab')2 and Fab fragments has been described in the literature since the beginning of the last century, in 1936 IA Parfentjev (patents no. US 2,065,196, US 2,123,198 and 1 US 2,175,090).Most methods are based on the use of the physicochemical and thermodynamic properties of proteins, such as their solubility, shape, and affinity. Consequently, in recent years, techniques have enabled the production of F(ab')2 and Fab antibody fragments, as in the case of US patents 5,733,742 by Landon and 4,849,352 by Sullivan et al. However, the current development of protein analysis technologies has become a tool for the development and improvement of enzymatically modified antibodies, as it has allowed for more in-depth characterization of immunogens used in the production of hyperimmune mammalian plasma, as well as the quantification of the neutralizing activity of specific antibodies and the control of the purification process.The production of modified antibodies (fragments) occurs when immunoglobulins (IgG) are enzymatically digested by various proteolytic enzymes such as pepsin or papain. In both cases, the Fc fragment is eliminated, but with pepsin, an F(ab')2 fragment is obtained, while with papain, two Fab fragments are produced. These F(ab')2 fragments retain the characteristics of complete antibodies with respect to their specificity, affinity, and stability. In addition to the absence of the Fc region of antibodies, the occurrence of adverse effects (such as, for example, anaphylactic reactions) is eliminated because the Fc region binds to various cellular receptors such as the Fc receptor and to other molecules of the immune system such as complement proteins, as well as other effector functions such as opsonization, cell lysis and degranulation of mast cells, basophils and eosinophils. Mexican patent MX230257, owned by J. Lépez de Silanes et al. of the Bioclon Institute, and its equivalent patents US Nos. 6,709,655, 7,485,303, and 8,075,893, describe a method for preparing a pharmaceutical composition comprising F(ab')2 fragments with unique characteristics acquired by the preparation method, which is free from: - Complete antibody molecules - Protein molecules of another nature - Albumin - Fibrinogen - Viral particles - Pyrogens. However, the scope of patent MX230257 and its US equivalents does not include all antivenom products. OBJECTIVE OF THE INVENTION: An objective of the invention relates to the production and development of a formulation of highly specific modified antibodies obtained from mammals.Another objective of the present invention relates to the production and development of a lyophilized formulation of highly specific modified antibodies, or variations thereof, from mammals. Another objective of the invention relates to the production and development of a highly specific, modified neutralizing formulation of antibodies from mixtures of heterologous proteins, peptides, and other organic and inorganic components having various specific activities, which may include, but are not limited to, venoms from venomous animals. A further objective of the present invention includes a useful production process for the hyperimmunization of horses for the production of highly specific antibodies, a modification (fragmentation) process, and a purification process.Another objective of the invention is to impart high purity and high specificity properties to the lyophilized injectable formulation. Other objectives and aspects of the present invention will be evident to individuals of ordinary competence upon examination of this disclosure. SUMMARY DESCRIPTION OF THE DRAWINGS Figure 1 illustrates two tables comparing the antivenoms of the present invention for the snake species B. apser and C. durissus with the antivenom of the prior art. The percentage of specific antibodies against the venom used in the immunization of antibody-producing horses is shown to illustrate what percentage of the immunoglobulin present in a vial of the used batches is related to the venoms. Figure 2 shows two tables comparing the antivenoms of the present invention for the snake species B. apser and C. durissus with the antivenom of the prior art.It gives the quantity in milligrams of antivenom required to neutralize one milligram of venom used in the immunization of antibody-producing horses. Figure 3. Electrophoresis during the conditioning phase of hyperimmune plasma before enzymatic hydrolysis. Column 1, molecular weight marker. Column 2, 0.5% albumin standard. Column 3, 1.0% albumin standard. Column 4, 3.0% albumin standard, 3% IgG + IgGT. Column 5, 5% IgG + IgGT. Column 6, 7% IgG + IgGT. Column 7, 10% IgG + IgGT. Column 8, Batch 1 of plasma conditioned before enzymatic hydrolysis. Column 9, Batch 1 of plasma conditioned before enzymatic hydrolysis. Column 10, Batch 1 of plasma conditioned before enzymatic hydrolysis. Figure 4. Graph showing the formation of F(ab')2 fragments during pepsin enzymatic digestion. Figure 5. Electrophoresis (SDS-PAGE) for three test lots after pepsin enzymatic digestion. Column 1, molecular weight marker. Column 2, 0.5% albumin standard. Column 3, 1.0% albumin standard. Column 4, 3.0% albumin standard, 3% IgG + IgGT. Column 5, 5% IgG + IgGT. Column 6, 7% IgG + IgGT. Column 7, 10% IgG + IgGT. Column 8, Lot 1 of plasma digested with pepsin for F(ab')2 fragment production. Column 9, Batch 2 of pepsin-digested plasma for the production of F(ab')2 fragments. Column 10, Batch 3 of pepsin-digested plasma for the production of F(ab')2 fragments. Figure 6. Precipitation step with ammonium sulfate electrophoresis SDSPAGE of three development batches after precipitation with ammonium sulfate.Using the salting-out technique (high salt concentration), the extraction of non-F(ab')2 proteins (such as pepsin, undigested fibrinogen, complete IgG, and the production of numerous peptides during digestion) is shown in a 35% (w / v) ammonium sulfate solution. Column 1, molecular weight marker. Column 2, 0.5% albumin standard. Column 3, 1.0% albumin standard. Column 4, 3.0% albumin standard, 3% IgG + IgGT. Column 5, 5% IgG + IgGT. Column 6, 7% IgG + IgGT. Column 7, IgG + IgGT at 10%. Column 8, first batch of supernatant after precipitation with ammonium sulfate. Column 9, second batch of supernatant after precipitation with ammonium sulfate. Column 10, third batch of supernatant after precipitation with ammonium sulfate. Also shown is a chromatogram obtained from a molecular weight exclusion HPLC analysis, demonstrating the supernatant composition at this stage, consisting of F(ab')2 fragments and low molecular weight components (less than 20 µDa). Figure 7. Depth filtration step. Electrophoretic (SD-SPAGE) and chromatographic (GE-HPLC) analysis of the clarified soluble phase, respectively. The results show a decrease in high molecular weight components (HMWC) to below 0.45%. SDS-PAGE electrophoresis of three development batches in the depth filtration step. Column 1, molecular weight markers.Column 2, 0.5% albumin standard. Column 3, 1.0% albumin. Column 4, 3.0% albumin. Column 5, 3% IgG + IgGT. Column 6, 5.0% IgG + IgGT. Column 7, 7% IgG + IgGT. Column 9, clarified Batch 2. Column 10, clarified Batch 3. Figure 8. Diafiltration stage. This step removes ammonium sulfate and most of the low molecular weight peptides present in the product, and equilibrates the isotonicity of the F(ab')2 fragments with 0.85% isotonic saline. At the end of this step, the product is concentrated to remove the solvent by forced diafiltration. The percentage composition results are shown during the diafiltration process. HPLC analysis of permeates resulting from the ultrafiltration step of the product in the process.a) Permeate for 0 diafiltrations, b) Permeate for 2 diafiltrations, c) Permeate for 4 diafiltrations, d) Permeate for 6 diafiltrations, e) Permeate for 8 diafiltrations, 0 permeate for 10 diafiltrations. Figure 9. SDS-PAGE electrophoresis for the three batches at the diafiltration stage, and HPLC analysis of the ultrafiltered product in the process. Column 1, molecular weight marker. Column 2, 0.5% albumin. Column 3, 1.0% albumin. Column 4, 3.0% albumin, 4.IgG + 3% IgGT. Column 5, ultrafiltered product from batch 1. Column 6, ultrafiltered product from batch 2. Column 7, ultrafiltered product from batch 1. Column 8, IgG + IgGT at 3.0%. Column 9, IgG + IgGT at 5.0%. Column 10, IgG + IgGT at 7.0%. DETAILED DESCRIPTION OF THE INVENTION For better understanding and comprehension, definitions of the terms used in the present invention are provided, but these definitions do not limit the scope of the invention. “Lyophilized injectable formulation.” This term refers to a lyophilized injectable pharmaceutical form that is sterile, free from contamination (physical, chemical, microbiological, and biological), with a high purity exceeding 85%, a total protein concentration not exceeding 5%, free from mammalian plasma components, and conforming to the concentration, quality, purity, safety, and potency specifications of the United Mexican Pharmacopoeia (FEUM) and the Sixth United States Pharmacopoeia (USP), for intramuscular or intravenous administration.“Modified Antibodies” This term refers to immunoglobulin G that is modified by removing the crystallizable fragment (Fc) and generating a bivalent fragment of type F(ab')2, which eliminates the risk of adverse effects and is highly specific for the antigenic complexes generated by the original immunoglobulins. “High Specificity” To achieve high-specificity immunotherapy, it is necessary that the hyperimmune plasmas, which are the material for those produced, have very high neutralizing titers. High plasma neutralizing titers force the antivenom to neutralize the same amount of venom with smaller amounts of antivenom, resulting in greater safety for the patient because they receive lower amounts of heterologous proteins. High plasma neutralizing titers are achieved from five main factors: (1) the use of certified venoms of the highest quality.(2) The use of immunization venoms obtained from a larger number of individuals of the same venomous species and from geographically different areas, for good coverage of the biochemical diversity of venom composition. (3) The rational and alternative use of adjuvants with different mechanisms of action. (4) The maturation of the humoral immune response over several months (at least six). (5) Optimal care and handling of the horses producing the hyperimmune plasma, including a balanced diet, exercise, friendly handling, and, very importantly, the performance of plasmapheresis (return of the red blood cell pack) within 120 minutes. 25 “Complex Antigens”.These are heterologous mixtures of proteins, peptides, and other organic and inorganic compounds, having specific activities and which may include, but are not limited to, arachnids, snakes, birds, fish, crustaceans, insects, frogs, anemones, and corals, among others. "Hypelimmunization." This term refers to the systematic process of inoculating (dosage, frequency, method of administration) an immunogenic mixture (which may be composed of proteins, peptides, and other organic and inorganic compounds) for the production of specific immunoglobulins. It also includes the methodology and experimental strategy for controlling and analyzing the specific neutralizing activity of the immunoglobulins.10 Methodology According to the preceding description, the first step consists of producing immunoglobulins through hyperimmunization with pre-treated and modified complex antigens from mammals such as horses, sheep, goats, and rabbits, among others, using specific detoxification methodologies such as, for example, gamma irradiation. This is followed by monitoring in vaccination systems to achieve maximum specificity. Next comes a modified antibody production process in which the Fc fraction is removed by enzymatic digestion using a proteolytic enzyme, which, in this case, is pepsin. 20 The manufacturing process consists of 9 steps. The first step is the dilution of plasma in three volumes of isotonic saline (0.85%) pre-treated with thimerosal, followed by adjusting the pH to 3.5–4.0 and the temperature to 18–20 °C.Subsequently, a pretreated and pre-activated acidic pepsin solution is added to obtain a final pepsin concentration of 0.9–1.1% (with at least 400 IU / g), and the conditions are maintained for 180 minutes to complete the removal of the Fc fraction of immunoglobulins (production of F(ab')2). This is verified by SDS-8 PAGE analysis, with a maximum IgG concentration of 2% and a maximum albumin concentration (the protein with the highest concentration in mammalian plasma) not exceeding 0.5%. At this stage, the enzymatic digestion process hydrolyzes the larger molecular proteins, such as albumin, fibrinogen, and other coagulation factors present in the hyperimmune plasma, converting them into small peptides with an approximate molecular weight of 15 kDa.A 50% aqueous ammonium sulfate solution is then added (previously filtered through a 0.2-micron cellulose filter) to eliminate the possibility of microbial contamination, in order to achieve a final saturation concentration of 35% at a temperature of 2 to 8 °C, with the aim of promoting the precipitation of low molecular weight components (below 15 kDa). Once the solution has precipitated, it is filtered through a modified cellulose fiber depth filtration module with a final nominal filtration gradient of 100 to 0.8 microns and conforming to the United States Code of Federal Regulations, Title 21, Sections 177.2260(e), (f), (g), (h), (i), (j), (k), and (l). The plastic components are included in the provisions of the United States Code of Federal Regulations, Title 21, Section 177.1520. The filtrate obtained is kept between 2 °C and 8 °C and the pH has been adjusted between 6.8 and 7.0. The filtrate was then diafiltered using tangential flow filtration equipment with a 50 kDa polyethersulfone membrane and borate buffer as the diafiltration buffer. This process is carried out continuously, maintaining a diafiltration separation of less than 1000 s⁻¹, which allows for the removal of ammonium sulfate used during precipitation as well as low molecular weight components (less than 15 kDa) that might be present after enzymatic digestion. The diafiltered product is collected in sterile, pyrogen-free containers and filtered through a filter with a pore size of 20 nanometers; this operation is called nanofiltration because of the nanometer-sized pores. The purpose of nanofiltration is to remove any remaining viral load.Once the product is nanofiltered, cryopreservation and isotonicity regulating agents are added in a unit operation called Formulation, in which mannitol (9 mg / mi), alanine (18 mg / mi), and polysorbate 80 (0.1 mg / mi) are added. Finally, the product is filtered in an aseptically classified area through a 0.1-micron PVDF filter to remove any potential microbial contamination, in an operation called final aseptic filtration. The formulated product is dispensed into Type 1 vials conforming to FEUM and USP Pharmacopoeia requirements. Dosage products under aseptic conditions are lyophilized under standard conditions (freezing to -40 °C for 8 hours and primary drying at -5 °C with a rate gradient of 5.2 °C / hour, and secondary drying at 45 °C for 10 hours at a rate of 9 °C / hour).These conditions allow for the removal of up to 98% of the water content, an advantage because, in addition to water removal, product stability is increased. Furthermore, this allows it to maintain its biological properties. The lyophilized product is evaluated through various tests, including leak tests, residual moisture tests, and a 100% visual inspection to determine the presence of particles, in addition to an analysis of all the established quality specifications for a lyophilized injectable product, which include sterility, safety, security, and neutralizing potency. The following examples are means of illustrating the invention, without limiting its scope. Example 1. Hyperimmunization of horses and production of highly specific immunoglobulins by the process of the present invention. Figure 1 shows two tables comparing the antivenoms of the present invention for the snake species *B. apser* and *C. durissus* with the prior art antivenom as a percentage. The experimental measurement was performed by affinity chromatography as follows: each venom was covalently coupled to cyanogen bromide-activated sepharose 4B particles. Affinity columns with the corresponding sepharose 4B-venom particles were mounted. 10 mg of each of the indicated antivenom batches were passed through the column. Non-specific immunoglobulins passed through the affinity column without interacting with the venom. Specific immunoglobulins (specific antibodies) bound to the venom in the column and eluted with 100 mM acetic acid. The amounts of non-specific immunoglobulins and specific antibodies were measured to calculate the percentage of specific antibodies.A higher percentage of specific antibodies means better quality antivenom, as it requires less protein to achieve the therapeutic effect and, consequently, increased antivenom safety. The antivenom product of the present invention (high-specificity immunotherapies) has, on average, 17.7% and 19.7% specific antibodies, respectively, for 15 of the venoms used in horse immunization. Antivenoms in the prior art have fluctuated between 4.3% and 13.8% specific antibodies for one snake species and between 3.7% and 10.1% for the other. The total amount of protein per vial is lower when the specific antibody level is higher, and vice versa. Example 2. Determination of the high specificity of immunoglobulins produced in hyperimmunized horses, using the method of the present invention.Figure 2 shows tables comparing the antivenom of the present invention for the snake species *B. apser* and *C. durissus* with the antivenom of the prior art. The quantity is given in milligrams of antivenom required to neutralize 11 milligrams of venom used in the immunization of antibody-producing horses. The experiment was designed using median effective doses (ED50) versus three median lethal doses (LD50), following the conditions and protocols described in Casasola et al., 2009. The ED50s are obtained in microliters of venom required to neutralize three LD50s, and since the protein concentration of the antivenom is known, the ED50s can be calculated in terms of protein quantities. The LD50s have their equivalent in mass of venom; therefore, the conversion to mg of antivenom required to neutralize one milligram of venom is straightforward. In this case, the fewer milligrams of antivenom required to neutralize one milligram of venom, the better the antivenom, since a smaller quantity is needed for the same efficacy. The high-specificity immunotherapies of the invention require, on average, 6.4 and 11.6 mg, respectively, to neutralize the two venoms used to immunize horses. Whereas the antivenom of the prior art requires between 9 and 49 mg to neutralize one of the species and between 18.2 and 34.9 mg for the other. Example 3. Evaluation and control of the production of modified antibodies (F(ab')2 fragments) by applying the process of the present invention.Figure 3 shows the SDS-PAGE analysis under reducing conditions (4% of the concentration gel and 12% of the separated gel) in the conditioning phase of the hyperimmune plasma prior to enzymatic hydrolysis, showing the composition of the hyperimmune plasma before the production process described in the present invention. The graph in Figure 4 illustrates the F(ab')2 fragment formation evaluated by SDS-PAGE and is compared to known concentration standards; this shows that, under the processing conditions, it is possible to achieve a yield of at least 95% F(ab')2 fragment formation.Figure 8 presents the results of the molecular exclusion HPLC analysis, which determines the percentage composition of Fab: F(ab')2, 5 dimers, soluble oligomers, and low molecular weight impurities, under appropriate mobile phase polarity conditions, using a support with specific molecular exclusion characteristics and a measurement system with a 280-nanometer UV detector. In Figure 8, the elution order is: soluble oligomers, dimers, F(ab')2, Fab, and low molecular weight components. The purity of the F(ab')2 fragments is greater than 85%. Example 4. Evaluation of the quality criteria of a lyophilized injectable pharmaceutical formula based on modified antibodies (F(ab')2 Fragments) in accordance with the quality standards of the FEUM (Pharmacope of the United Mexican States) and the USP (American Pharmacopoeia 15).The process for producing highly specific immunotherapies based on F(ab')2 fragments, as described in this invention, is carried out in accordance with Good Manufacturing Practices (GMP), which are guidelines for obtaining industrial-grade products that comply with the guidelines specified in FEUM and USP. These guidelines define the key properties of purity, safety, concentration, identity, and potency to be maintained for products obtained from the described process. The examples above are provided for illustrative purposes only and are not intended to limit the scope or content of the invention. The invention is described in more detail with reference to the claims set forth below.

Claims

DEMANDS 1. A process for preparing a composition of highly specific modified F(ab')2 type antibodies, obtained from IgG immunoglobulins of hyperimmune horse plasma immunized with venom, characterized in that said process comprises the following steps: a) dilute the plasma in three volumes of an isotonic saline solution at 0.85%, pretreated with thimerosal, followed by an adjustment of the pH conditions between 3.5 and 4.0, at a temperature between 18°C ​​and 20°C, b) add a pretreated and pre-activated acidic pepsin solution to the conditioned plasma obtained in step a), in order to obtain a final pepsin concentration of between 0.9% and 1.1%, maintaining the pH and temperature conditions of step a) for 180 minutes in order to obtain an enzymatic digestion product, c) add to the enzymatic digestion product of step b) a 50% aqueous solution of ammonium sulfate, at a final saturation concentration of 35% and at a temperature between 2°C and 8°C, in order to obtain a precipitated solution, d) filter the supernatant of the precipitated solution obtained in step c) through a depth filtration module of modified cellulose fibers, with a final nominal filtration gradient of 100 to 0.8 microns, in order to obtain a filtrate, e) Store the filtrate obtained in step d) at a temperature between 2°C and 8°C, at a pH of 6.8 to 7.0, and follow with a diafiltration period on a tangential flow filtration fluid using a 50kDa polyethersulfone membrane and borate buffer as the diafiltration buffer, continuously maintaining a diafiltration separation of less than 1000 s⁻¹ between the tangential flow filtration membrane and the proteins; furthermore, the isotonicity equilibrium of the F(ab')₂ fragments is made with an isotonic saline solution of 0.85%, in order to obtain a diafiltered product, f) recover the diafiltered product obtained in step e) then perform its nanofiltration through a filter with a pore size of 20 nanometers.

2. A method according to claim 1, characterized in that at step b), the maximum concentration of IgG is 2% and the maximum concentration of albumin is not greater than 0.5% according to an analysis by SDS-PAGE electrophoresis.

3. Composition of highly specific antibodies capable of being obtained using the preparation process according to claim 1 or 2, characterized in that it has a purity greater than 85% in F(ab')2 fragments.

4. Composition according to claim 3, characterized in that the F(ab')2 fragments are neutralizers of heterologous mixtures of proteins, peptides and other organic and inorganic components present in the venom of selected biological species in the group consisting of: snakes, scorpions, spiders, microorganisms, molluscs, fish, frogs, insects, anemones, corals or combinations thereof.

5. Antibody composition according to claim 3 or 4, characterized in that it is in the form of a lyophilized injectable formulation.

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