TR202021936A2 - Production of Hyperimmune Antiserum for Therapeutic Purposes Against COVID-19 - Google Patents
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Abstract
Bu buluşta hayvan denemelerinden sonra elde edilen immunglobulinler kimliksizleştirilerek tüm canlılarda kullanılacak hale getirilmiştir. Bu buluş özelinde insanlarda kullanıma uygun olarak saflaştırılmıştır ve antiserumlar elde edilmiştir. Bu buluş ile atlarda ve tavşanlarda yüksek miktarda antikor oluşturduğu ve bu hayvanların buluşa konu olan yöntem ile yapılan anti serum (hiperimmun serum) çalışmalarında rahatlıkla kullanılabileceği kanıtlanmıştır. Böylece insanlara dışarıdan antikor vererek mevcut antikor miktarı artırılacak ve daha hızlı ve az maliyet ile iyileşme sağlanacaktır.In this invention, the immunoglobulins obtained after animal experiments were de-identified and made to be used in all living things. In particular, this invention was purified for use in humans and antisera were obtained. With this invention, it has been proven that high amounts of antibodies are formed in horses and rabbits and that these animals can be used easily in anti-serum (hyperimmune serum) studies performed with the method of the invention. Thus, by giving antibodies to people from outside, the amount of available antibodies will be increased and recovery will be provided faster and with less cost.
Description
TARIFNAME COVI D-l9”a Karsi Terapötik Amaçli Hiperimmun Antiserum Üretimi Bulus, insanlarda tedavide kullanilmak üzere ETLVET-3 susundan elde edilmis COVID-19 inaktif virusu enjekte edilmis atlardan saflastirilan antikorlarin elde edildigi biyolojik ürün üretimi ile ilgilidir. DESCRIPTION Production of Hyperimmune Antiserum for Therapeutic Purposes Against COVI D-19 The invention is a COVID-19 strain derived from the ETLVET-3 strain for use in the treatment of humans. Biological product from which purified antibodies are obtained from horses injected with inactive virus relates to production.
Teknigin bilinen durumu COVID-19 tedavisi için atlar tarafindan üretilen at menseili antikorlarin, bazi durumlarda, hastaliga neden olan viruslari nötralize etmek için 40 ila 100 kata kadar daha fazla güce sahip oldugu gösterilmistir. Bu durum, hayvanlar tarafindan üretilen antikorlarin COVID-19 virusunu "serumlarin son ürün haline ulastiginda bile" 100 kata kadar daha fazla güçle nötralize ettigi anlamina gelir. SARS, MERS ve COVlD-l9 için hali hazirda etkili bir ilaç ya da antiserum bulunmamaktadir. SARS”a karsi at hiperimmun serumu hazirlanmasi ve gelistirilmesi üzerine çalismalar bulunmaktadir. Hayvan Coronavirus enfeksiyonlarina karsi immunglobulin kullaniminin etkili bir yol oldugunu gösteren birçok çalisma oldugu bildirilmis ve bu çalismalardan yola çikarak COVlD-l9”a spesifik hiperimmun globulinlerin SARS COV-Z için pasif bagisiklik yanit amaciyla destekleyici bir tedavi olabilecegi ifade edilmistir. Yapilan bir çalismada at anti serumu, kuduz gibi baska birçok hastaligin tedavisinde kullanildigi bilindigi için tercih edilmistir. At hiperimmun globülini SARS COV- 2 enfeksiyonu kontrolünde kullanilabilecek bir Stratejik ajan olarak düsünülmüstür. COVID- 19”a spesifik hiperimmun globülinlerin eldesi pahali bir yöntemdir. Her yerde uygulanamaz. State of the art Equine antibodies produced by horses for the treatment of COVID-19, in some cases, 40 to 100 times more powerful to neutralize disease-causing viruses has been shown. This is because antibodies produced by animals to COVID-19 virus with up to 100 times more power "even when the serum reaches the final product" It means neutralized. An already effective drug for SARS, MERS and COVID-19 or no antisera. Preparation of equine hyperimmune serum against SARS and There are studies on its development. Against animal Coronavirus infections There are many studies showing that the use of immunoglobulin is an effective way. have been reported and based on these studies, hyperimmune globulins specific for COVID-19 It is stated that it can be a supportive treatment for SARS COV-Z for passive immune response. has been made. In a study, equine antiserum was found to be effective against many other diseases such as rabies. It is preferred because it is known to be used in the treatment of Equine hyperimmune globulin SARS COV- It has been considered as a strategic agent that can be used in the control of 2 infections. COVID- Obtaining 19-specific hyperimmune globulins is an expensive method. It cannot be applied everywhere.
Baska bir çalismada elde edilen canli SARS Coronavirus izolatlari, atlarin immunizasyonu için kullanilmistir. Atlar dört defa immunize edilmistir. Bu amaçla 4-9 yas araligindaki saglikli atlar seçilmistir. Belirli araliklarla alinan kan örnekleri antikorlar yönünden degerlendirmis ve antikor titresinin en yüksek seviyeye 8 haftalik periyot sonunda ulastigi tespit edilerek plazma örnekleri toplanmistir. Elde edilen örneklerden lgGller diyaliz ile toplanmis ve IgG”lerin kesim islemini takiben elde edilen F(ab”)2 parçalari ultrafiltrasyon yöntemi ile saflastirilmis ve SDS-PAGE ile protein safligi yönünden kontrol edilmistir. Elde edilen sonuçlar SARS-COV F69 susu ile immunize edilen saglikli hayvanlarda etkin düzeyde, Spesifik ve nötralizan antikor üretiminin gerçek]estirilebilecegini göstermistir. Live SARS Coronavirus isolates obtained in another study, immunization of horses has been used for. Horses were immunized four times. For this purpose, between the ages of 4-9 healthy horses are selected. Blood samples taken at regular intervals for antibodies evaluated and the antibody titer reached its highest level at the end of the 8-week period. plasma samples were collected. IgGls from the obtained samples were obtained by dialysis. collected and the F(ab”)2 fragments obtained after cutting the IgGs by ultrafiltration. method and checked for protein purity by SDS-PAGE. in hand The results obtained were effective in healthy animals immunized with the SARS-COV F69 strain. demonstrated that production of specific and neutralizing antibodies could be achieved at a high level.
Heterojen ve/veya üst düzeyde saflastirilmamis antiserum kullaniminin Ciddi anafilaktik sok gibi yan etkileri bulunmaktadir. Bu yan etkileri en aza indirmek için SARS-CoV-2”ye karsi olusan lgG”ler attan gelecek FC parçalarini elimine etmek amaciyla Spesifik antikor yapisini bozmayacak sekilde pepsin ile muamele edilerek F(ab”)2 parçalari elde edilmis ve anyon- degisim kromatografi yardimiyla % 90 saflastirilmistir. Severe anaphylactic shock from the use of heterogeneous and/or highly purified antiserum has side effects such as Against SARS-CoV-2 to minimize these side effects The IgGs that are formed have the specific antibody structure in order to eliminate the FC fragments that will come from the horse. F(ab”)2 fragments were obtained and anion- 90% purified by exchange chromatography.
Benzer bir tErapötik antiserum çalismasi da Ebola virusuna karsi yapilmistir. Çalismada patojen Ebola virusu yerine rekombinant modifiye Vaccinia Ankara (MVA') virus tarafindan sentezlenen virus benzeri parçaciklar (VLP”s) virusun de novo eksprese edilmis glikoprotein yapilarina karsi adjuvan varliginda tavsanlarda hiperimmun serum eldesi için kullanilmistir. A similar therapeutic antiserum study was also conducted against Ebola virus. in the study by recombinant modified Vaccinia Ankara (MVA') virus instead of the pathogenic Ebola virus. Synthesized virus-like particles (VLP's) de novo expressed glycoprotein of the virus It has been used to obtain hyperimmune serum in rabbits in the presence of adjuvant against their structures.
Sonuçta virusun orijinal konformasyonuna sadik kalan protein yapilarinin (VLP yapilari) enfeksiyona karsi daha yüksek oranda ve etkili immun yanit olusturdugu saptanmistir. As a result, protein structures (VLP structures) that remain faithful to the original conformation of the virus It has been determined that it has a higher rate and effective immune response against infection.
Ayrica bu Çalismada ortaya konulan yaklasim ile Nipah virusuna karsi yapilan hiperimmun globulin üretim denemelerinde umut verici sonuçlar elde edilmesi, bu yöntemlerin baska viruslara da transfer edilebilecegini göstermistir. In addition, with the approach presented in this study, hyperimmune tests against Nipah virus were made. Obtaining promising results in globulin production trials, these methods It has been shown that it can also be transferred to viruses.
Immünoterapi, enfeksiyöz ve non-enfeksiyöz hastaliklar için kullanima hazir antikorlarla güçlü bir tedavi araci haline gelmistir. Her ne kadar influenza virusu için asi ve ilaç seçenekleri olsa da virusun hizla mutasyona ugrayan anjtijenik yapisi yüzünden mevcut tedaviler gün geçtikçe daha az etkili olmaya baslamistir. Bu durum özellikle yüksek risk altinda olan bireyler için alternatif tedavi seçeneklerinin bulunmasini gerekli kilmaktadir. Immunotherapy with ready-to-use antibodies for infectious and non-infectious diseases It has become a powerful therapeutic tool. Although vaccine and medicine for influenza virus Although there are options, it is available due to the rapidly mutating angitigenic nature of the virus. treatments are becoming less and less effective day by day. This is particularly high risk. It makes it necessary to find alternative treatment options for individuals who are under.
Hiperimmun globülin eldeSi için serum örnekleri disinda baska kaynaklari da kullanmaya yönelik çalismalar mevcuttur. Bir çalismada farkli bir insan Rotavirus (HRoVJ genotipine karsi, konvansiyonel sigir rotavirus susu (B RoVi ile asilanmis ineklerden alinan hiperimmun bovin kolostrumunun (H BC) koruyucu etkisini gösteren veriler sunulmustur. Konvansiyonel BROV asisinin, sigir kolostrumunun anti-insan rotavirusuna karsi koruyucu etkinligi arttirmak için yeterli oldugu gösterilmis ve böylece fonksiyonel bir gida olarak hiperimmun sigir kolostrumunun üretilmesi için yeni bir yaklasim ortaya konulmustur. Bu çalismaya temel teskil eden benzer bir çalisma farelerde gerçeklestirilmistir ve Influenza virusu için yapilmistir. Çalisma sonunda elde edilen ürünün, günlük uygulamaya ihtiyaç duyulmamasindan dolayi antiviral ajanlarin kullanimina göre önemli avantajlara sahip oldugu ve ilaca dirençli viruslara karsi tercih edilen bir kontrol önlemi olabilecegi belirtilmistir. Daha önce asilanmamis veya yeterli serokonversiyon gelistirememis asilanmis bireylerde, özellikle yaslilarda veya bagisiklik sistemi zayif olanlarda, poliklonal antikorlar ile tedavinin, morbidite ve mortaliteyi azaltmada önemli rol oynayabilecegi vurgulaninistir. To obtain hyperimmune globulin, it is necessary to use other sources besides serum samples. studies are available. In one study, a different human Rotavirus (HRoVJ genotype) against the conventional bovine rotavirus strain (hyperimmune strain from cows vaccinated with B RoVi) Data showing the protective effect of bovine colostrum (H BC) are presented. conventional Protective efficacy of BROV vaccine against anti-human rotavirus of bovine colostrum has been shown to be sufficient to increase hyperimmune A new approach has been introduced for the production of bovine colostrum. to this study A similar substantive study was carried out in rats and for Influenza virus has been made. The product obtained at the end of the study needs daily application. It has significant advantages over the use of antiviral agents because it is not heard of. and may be a preferred control measure against drug-resistant viruses. specified. Previously vaccinated or vaccinated who did not develop adequate seroconversion polyclonal antibodies in individuals, especially the elderly or those with compromised immune systems It has been emphasized that treatment with drugs can play an important role in reducing morbidity and mortality.
Corona virus ile ilgili olarak yapilan immunoterapi çalismalari ile ilgili olarak su an için COVID-19“a karsi gelistirilmis bir immunoterapi bulunmamakla beraber Çin°de klinik deneyler için basvurusu yapilmis çalismalar da mevcuttur. Bu çalismalardan birinde intravenöz immünoglobulinin (IVIG) pasif bagisiklik, antienflamatuar ve immünomodülatör etkisinin son on yilda yapilan çalismalarla kanitlandigini, siddetli enfeksiyonun tedavi etkinligini ve prognozunu iyilestirilebildigi belirtilmistir. Buradan yola çikarak klinik çalismalar baslatilmis ve siddetli veya kritik derecede 2019-nCoV solunum hastaligi olan insanlarda IVIG tedavisinin etkililiginin ve güvenliliginin denenmesi planlanmistir. Regarding the immunotherapy studies on Corona virus, for now Although there is no immunotherapy developed against COVID-19, there is no clinical practice in China. There are also studies that have been applied for experiments. In one of these studies passive immune, anti-inflammatory and immunomodulatory of intravenous immunoglobulin (IVIG) its effect has been proven by the studies carried out in the last ten years, the treatment of severe infection It has been stated that its effectiveness and prognosis can be improved. From here, clinical studies have been initiated and patients with severe or critical 2019-nCoV respiratory disease It is planned to test the efficacy and safety of IVIG treatment in humans.
At serumu ile ilgili patent arastirmasi da yapilmis ancak karsilasilan patent baswrularinda bu 'bulus konusu ile ilgili bir açiklamaya rastlanilmamistir. tedavisi ve önlenmesi için kullanilan at serumu üretimi ile ilgilidir. Patent research on horse serum has also been carried out, but in the patent applications that have been met, No explanation has been found regarding the subject of this invention. It relates to the production of horse serum used for the treatment and prevention of
CN102335198 numarali patent basvurusu; kene kaynakli ensefalit VIrUSuna karsi at antiserumu hazirlama yöntemi ile ilgilidir. Patent application numbered CN102335198; against tick-borne encephalitis VIrUS relates to the method of preparing the antiserum.
CN103341163 numarali patent basvurusu; kuduz ve tetanoz için kullanilan çift etkili at antiserumu ve bunlarin hazirlama yöntemi ile ilgilidir. Patent application CN103341163; double-acting horse used for rabies and tetanus antiserum and their preparation method.
Bulusun çözümünü amaçladigi teknik problemler COVid-l9°un hayvanlarda özellikle atlarda ve tavsanlarda antikor yanit olusturup olusturmadigi bilinmemekteydi. Bu bulus ile atlarda ve tavsanlarda yüksek miktarda antikor olusturdugu ve bu hayvanlarin bulusa konu olan yöntem ile yapilan anti serum (hiperimmun serum) çalismalarinda rahatlikla kullanilabilecegi kanitlanmistir. Böylece insanlara disaridan antikor vererek mevcut antikor miktari arttirilacak ve daha hizli iyilesme saglanacaktir. Bulusa konu olan anti serumun piroj enite ve buna ek olarak zararsizlik testleri yapilmis ve basari elde edilmistir. Bu bulus kapsaminda virus izolasyonu, yüksek miktarda üretilmesi ve inaktivasyonu islemleri Biyogüvenlik Seviyesi 3 (BSL3) olan laboratuvarda tüm gerekli yasal ve etik izinleri alinarak yapilmistir. Deneinelerde kullanilan virus hayvanlarda hastalik yapmayacagi için (virus inaktive oldugu için) refahlarinda bir bozulma olmadan yasamlarina devam edeceklerdir. Technical problems that the invention aims to solve Antibody response to COVID-19 in animals, especially horses and rabbits was not known. With this invention, high amounts of antibodies in horses and rabbits anti-serum (hyperimmune) produced by these animals and made by the method subject to the invention. serum) has been proven to be easily used in studies. So that people By giving antibodies externally, the amount of available antibodies will be increased and a faster recovery will be achieved. will be provided. Pyrogenicity and harmlessness tests of the antiserum subject to the invention done and successful. Within the scope of this invention, virus isolation is production and inactivation processes in a Biosafety Level 3 (BSL3) laboratory All necessary legal and ethical permissions have been obtained. Virus used in experiments Since they will not cause disease in animals (since the virus is inactivated), their welfare will not deteriorate. They will go on with their lives without it.
Günümüzde kullanilan birçok antiviral hastaligin yan etkisi olmakta ve COVID-19 tedavisinde de kullanilan birçok ilaç yan etki göstermektedir. Oysa bu bulusa konu olan yöntem ile gelistirilen antiserumun hiçbir yan etki göstermedigi bulus kapsaminda yapilan piroj enite ve zararsizlik testleri ile Avrupa Farmakopesi °ne göre kanitlanmistir. Many antiviral diseases used today have side effects and COVID-19 Many drugs used in the treatment also show side effects. However, the subject of this invention Within the scope of the invention, the antiserum developed by the method did not show any side effects. It has been proven by pyrogenicity and harmlessness tests according to the European Pharmacopoeia.
Bulusun açiklamasi Bu bulusta hayvan denemelerinden sonra elde edilen immunglobulinler kimliksizlestirilerek tüm canlilarda kullanilacak hale getirilmistir. Bu bulus özelinde insanlarda kullanima uygun olarak sailastirilmistir ve antiserumlar elde edilmistir. Description of the invention In this invention, immunoglobulins obtained after animal trials are de-identified and made to be used in all living things. This invention is specifically suitable for use in humans. and antisera were obtained.
Sekilleriii Açiklamasi Sekil 1: SBS Page analizi sonucu. Description of Shapes Figure 1: SBS Page analysis result.
Sekil 2: MALDI TOF analizi sonucu. Figure 2: MALDI TOF analysis result.
Sekil 3: SARS-COV-Z virus antiserumundan (A) Nr ve (B) R kosullarda hazirlanan örneklerle yapilan % 8-16 SDS-PAGE analizi sonuçlari. Orijinal antiserum 1:10, 1:50 ve 1:100 oranlarinda seyreltilerek yüklenmistir. M: moleküler agirlik. Figure 3: SARS-COV-Z virus antiserum prepared under (A) Nr and (B) R conditions. 8-16% SDS-PAGE analysis results with samples. Original antiserum 1:10, 1:50 and It is loaded by diluting at 1:100 ratios. M: molecular weight.
Bulusun detayli açiklamasi: SARS COV-2 izolasyonu amaciyla hasta insanlardan alinan sürüntü örneklerinden RNA ekstraksiyonu (QlAamp cador Pathogen Mini Kit) yapilmistir. Dünya Saglik Örgütü”nün tavsiye ettigi primer dizinleri kullanilarak QuantiNova Pathogen + IC Kiti ile Biorad CFX- 96 cihazinda Real Time RT-PCR yöntemiyle COVID-l9 testi yapilmistir. Pozitif örneklerin Vero E6 hücre kültürüne ekimi gerçeklestirilmistir. Her gün sitopatolojik etki yönünden hücreler invert mikroskopta kontrol edilmistir. Hücrelerde dökülmeler görülünce -SOOC'de dondurulmustur. Sonrasinda hücreler 37°C°de çözülerek 4000 rpmide 15 dakika santrifüj edilmistir. Supernatantlar toplanmis ve kriyotüplere porsiyonlanarak bir kismi tekrar hücre kültürüne ekim, virus titrasyon ve ekstraksiyon amaciyla ayrilmistir. Geriye kalanlar ise - 80°C”ye kaldirilmistir. Ekstraksiyon yapilanlara yukarida bildirdigi sekilde Real Time RT- PCR testi uygulanmistir. PozitiI` tespit edildikten sonra Virus titrasyon testi yapilmistir. Bu amaçla virus loglO tabaninda sulandirilmistir. Önceden hazirlanmis 96 gözlü hücre kültürü pleytlerine virus sulandirmalari eklenmis ve bunlarin da üzerlerinde Vero E6 hücreleri eklenmistir. Virus sulandirmalarinda tespit edilen sitopatolojik etkilere göre degerlendirme yapilmistir. Spearman-Karber metoduna göre virus titresi hesaplanmistir. Yapilan eki m lerde bu titreye göre hesaplamalar gerçeklestirilmistir. Sonrasinda hücre kültüründe vims üretmek amaciyla da yukarida bildirilen yöntem uygulanmistir. Istenilen Virus hacmine ulasilincaya kadar bu adimlar tekrarlanmistir. Sonrasinda viruslar 0,2 um filtrelerden süzülmüstür. lnaktivasyon amaciyla Binaryetilenamin (BEI), formaldehit (FA) ve isil yöntemleri kullanilmistir. FA yönteminde FA orani 1:1000 olarak ayarlanmis ve III hacminde; B E I +FA yönteminde FA orani 122000 ve BEI hacimce %3 v/W oranda olacak sekilde eklenmistir. Detailed description of the invention: RNA from swab samples from sick humans for SARS COV-2 isolation extraction (QlAamp cador Pathogen Mini Kit) was done. of the World Health Organization Biorad CFX- with QuantiNova Pathogen + IC Kit using recommended primer sequences COVID-19 test was performed with Real Time RT-PCR method on 96 devices. of positive samples It was cultivated in Vero E6 cell culture. Every day in terms of cytopathological effect Cells were checked under an inverted microscope. When spills are seen in the cells -SOOC has been frozen. Afterwards, the cells were thawed at 37°C and centrifuged at 4000 rpm for 15 minutes. has been made. Supernatants were collected and portioned into cryotubes and some of them were re-celled. culture, the virus was separated for titration and extraction. The rest are - It has been raised to 80°C. As reported above, Real Time RT- PCR test was applied. Virus titration test was performed after positive detection. This For this purpose, the virus was diluted on a log10 basis. Pre-prepared 96 well cell culture Virus dilutions were added to the plates and Vero E6 cells were added on them. has been added. Evaluation according to cytopathological effects detected in virus dilutions has been made. The virus titer was calculated according to the Spearman-Karber method. In the plantings made Calculations were made according to this titer. To subsequently produce vims in cell culture For this purpose, the above-mentioned method was applied. until the desired virus volume is reached. These steps are repeated until The viruses were then filtered through 0.2 µm filters. Binaryethyleneamine (BEI), formaldehyde (FA) and thermal methods for inactivation used. In the FA method, the FA ratio was set as 1:1000 and in III volume; B E I +FA In the method, the FA ratio was 122000 and the BEI was added at a 3% v/W ratio by volume.
Kimyasallarin nötralizasyonu için son ürüne sodyum tiyosülfat (%2) eklenmistir. Viruslar 1 saat tutulmustur. Tüm bu islemler boyunca düzenli olarak numuneler alinarak Virus inaktivasyon kinetigi olusturulmustur. Inaktivasyonlarin dogrulanmasi amaciyla hücre kültüründe üç kör pasaj yapilmistir. Hücrelerde sitopatolojik etki görülmemesi ile Virus inaktivasyonUn dogrulanmasi saglanmistir. Sodium thiosulfate (2%) was added to the final product for the neutralization of chemicals. viruses It has been held for 1 hour. During all these processes, samples were taken regularly and the Virus inactivation kinetics was established. To confirm the inactivations, the cell Three blind passages were made in the culture. Virus with no cytopathological effect in cells Confirmation of inactivation is provided.
Nötralizasyon testi amaciyla 96 gözlü hücre kültürü pleytleri kullanilmistir. Serumlar logz tabanina göre sulandirilinistir. Üzerlerine lOODKlDso oraninda sulandirilmis SARS CoV-2 eklenmistir. Bir saat hücre kültürü etüvünde inkubasyona birakilmistir. Sonrasinda üzerlerine Vero E6 hücre kültürü süspansiyonu eklenmistir. Virus kontrol gözlerinde olusan sitopatolojik etki, hücre kontrol ve ters titrasyon sonuçlari olumlu çikinca test degerlendirilmistir. Antikor titreleri hesaplanmis ve atlarda istenilen yüksek antikor titresine ulasildiginda atlardan kan toplama asamasina geçilmistir. Bu plazmaferez asamasinda plazmaferez cihazi yardimi ile atlardan elde edilen kanlarin plazmalari ayrilmistir. Bu plazmalarin ekstra ajanlar yönünden kontrolleri Dünya Saglik Örgütü°nün belirledigi sekilde yapilmis ve uygun sonuçlarin elde edilmesi Sonrasi plazma saflastirma basamagina geçilmistir. Cell culture plates with 96 wells were used for neutralization test. Serums logz diluted according to the base. SARS CoV-2 diluted with LOODKlD50 on them has been added. It was incubated in a cell culture oven for one hour. After that Vero E6 cell culture suspension was added on them. If you have virus control eyes When the cytopathological effect, cell control and reverse titration results are positive, the test has been evaluated. Antibody titers were calculated and the desired high antibody titer was achieved in horses. When it is reached, the stage of collecting blood from the horses has been passed. At this stage of plasmapheresis Plasma of blood obtained from horses was separated with the help of plasmapheresis device. This Controls of plasmas for extra agents are as determined by the World Health Organization. plasma purification step after has been passed.
Toplanan plazmalara FTS eklenmistir. HCI asitle pH 3,3 yapilmis ve sicaklik 29°C”ye ayarlanmistir. 15 dakika beklenilerek %2°lik Pepsin (Sigma) solüsyonu hazirlanmis, 1 saat süreyle karisima eklenmistir. Sonrasinda %2-5°lik kaprilik asit ürüne 1 saatte oldukça yavas karistirilarak eklenmistir. Filtrelerden geçirilmis ve santritüj edilmistir. Baska bir fermentatöre alinarak pH 7`ye, sicaklik 56°C”ye getirilmistir. 30 dakika beklenerek 1 saat süre ile kuvvetliee karistirilmistir. Daha sonra ürün baska fermentatöre tiltrelerden geçirilerek alinmistir. FTS eklenip konsantre edilerek ultratiltrasyon asamasi gerçeklestirilmistir. 4200 rpng'de 2 saat süreyle santriûij edilmistir. Sonrasinda ürün kapsül filtreden geçirilerek bulusa konu olan final (son) ürün elde edilmistir. FTS was added to the collected plasmas. pH 3.3 was made with HCl acid and the temperature was set to 29°C. is set. 2% Pepsin (Sigma) solution was prepared by waiting for 15 minutes, 1 hour time was added to the mix. Afterwards, 2-5% caprylic acid is added to the product very slowly in 1 hour. added by mixing. It was filtered and centrifuged. Another It was taken to the fermenter and brought to pH 7 and temperature 56°C. 1 hour by waiting 30 minutes mixed strongly over time. Then the product is transferred to another fermenter from the filters. passed through. Ultrafiltration step by adding FTS and concentrating has been carried out. It was centrifuged at 4200 rpng for 2 hours. product after The final product, which is the subject of the invention, is obtained by filtering the capsule.
Bu asainalardan sonra elde edilen siselenmis son üründe sterilite, safsizlik, zararsizlik ve Pirojenite Testleri uygulanmistir. Sterilite ve endotoksin kontrolleri için besi yerleri kullanilmistir. Safsizlik testlerinde kromotografik ve SDS-PAGE yöntemlerinden elde edilen sonuçlar degerlendirilmistir. Pirojenite testlerinde Avrupa Farmokepesi'nde tavsiye edildigi sekilde tavsanlar kullanilarak sicaklik degisimleri takip edilmistir. Zararsizlik testlerinde Swiss albino fareler ve kobaylar kullanilmistir. Bu testlerde de Avrupa Farmokepesi”nde bildirilen agirlik artislari kontrol edilmistir. Son üründe önemli patojenler yönünden kontroller tekrar yapilarak Dünya Saglik Örgütü tarafindan bildirilen herhangi bir patojen Olup olmadigi arastirilmistir. Siselenmis son üründe bulunan protein miktarinin Avrupa Farmokepesi°nde bildirilen sinirlar içinde olup olinadigi kontrol edilmistir. After these steps, sterility, impurity, harmlessness and harmlessness in the bottled final product obtained. Pyrogenicity Tests were applied. Media for sterility and endotoxin controls used. Obtained from chromatographic and SDS-PAGE methods in impurity tests. the results have been evaluated. Recommendation in the European Pharmacopoeia on pyrogenicity tests Temperature changes were followed using rabbits as shown. Harmlessness Swiss albino mice and guinea pigs were used in the tests. In these tests, the European The weight gains reported in the "Farmokepesi" were controlled. Important pathogens in end product Controls were made again in terms of It has been investigated whether it is pathogenic or not. The amount of protein in the bottled final product It has been checked whether it is within the limits reported in the European Pharmacopoeia.
Yapilan tüm testler Avrupa Farmokepesi°ne uygun olmasi yönünden degerlendirilmis ve insan sagligina zararli olmadigi uluslararasi standartlara göre dogrulanmistir. All tests have been evaluated in terms of compliance with the European Pharmacopoeia and It has been verified according to international standards that it is not harmful to human health.
Son Ürün Kontrol Testleri Sterilite, Saßizlik ve Pirojenite Testleri: Yukarida asamalariyla açiklanan son üründe TSBI TCM, SABO besi yerlerinde 37°C ve °C'de sterilite ve LAL endotoksin kontrolleri Avrupa Farmakopesine uygun olarak yapilmistir (Ph. Eur. 2.6.1: Supp.10.1). Ayrica son üründe yapilan ekimlerde herhangi bir bakteriyel üremeye rastlanmaz iken “Safsizlik” kontrolleri kromotografik ve SDS-PAGE yöntemleri ile arastirilmistir. Final Product Control Tests Sterility, Illness and Pyrogenicity Tests: In the final product described above, TSBI TCM is stored at 37°C and SABO media. Sterility and LAL endotoxin controls at °C in accordance with European Pharmacopoeia (Ph. Eur. 2.6.1: Supp.10.1). In addition, any planting in the final product “Impurity” controls were chromatographic and SDS-PAGE while bacterial growth was not found. investigated with methods.
Son ürün Pirojenite deneyleri Avrupa Farmakopesi 10.0 kismina göre yapilmistir. üründen enjeksiyon yapilmistir. Son ürün olarak hem saf hali hem de 113 oraninda PBS ile sulandirilmis hali kullanilarak kontrol grubu dahil toplam üç grup yapilmistir. Her grubun kendi içinde homojen dagilim gösterdigi kontrol edildikten sonra birinci. üçüncü, besinci ve yedinci günlerde ad libutum olarak beslenen ve su içen farelerin agirlik artislari p<0,05 düzeyinde arastirilmistir. General Lineer Model repeated measures yöntemi ile zaman ve grup farki açisindan degerlendirilmistir. Olusan farkliliklar iki yönlü Dunnett testi ile saf ve 1/ 3 sulandirilmis gruplar kontrol grubuna karsi degerlendirilmistir. Final product Pyrogenicity tests were carried out according to European Pharmacopoeia part 10.0. The product was injected. As the final product, both in its pure form and with PBS at the rate of 113 A total of three groups, including the control group, were made using its diluted form. Each group the first after it was checked that it showed homogeneous distribution within itself. third, fifth and Weight gains of rats fed ad libutum and drank water on the seventh day p<0.05 level was investigated. General Linear Model repeated measures with time and evaluated in terms of group difference. The differences were determined by the two-way Dunnett test. 1/3 diluted groups were evaluated against the control group.
Son Ürünün Ekstra Ajanlar Yönünden Testi: Bakteriyoloji, kuduz, tüberküloz, paratüberküloz ve ruam teshis, Viral teshis, spiroket hastaliklari, yetistirme hastaliklari ve parazitoloji laboratuvarlari tarafindan zoonoz ve atlarda bulunabilecek ajanlar yönünden kontroller yapilmistir. Test of the End Product for Extra Agents: Bacteriology, rabies, tuberculosis, paratuberculosis and rumen diagnosis, Viral diagnosis, spirochete diseases, breeding diseases and parasitology laboratories by zoonosis and Controls were made in terms of agents that may be present in horses.
Protein Miktar Tayini: Protein miktar tayini çesitli yollarla yapilmis olsa da nihai tayin Leco FP 521 model cihaz ile Dumas metoduna bagli kalarak 95006& ürünün yakilmasi ve ortaya çikan azotun yüzde degeri elde edilmistir. Determination of Protein Quantity: Although protein quantitation has been done in various ways, the final determination is Leco FP 521 model device. Combustion of 95006& product, adhering to the Dumas method, and the percentage of nitrogen produced value is obtained.
Plazma ve Son Ürün Testleri: 75 L olarak toplanan plazmanin konsantrasyon ve saflastirrna islemini takiben yapilan virus nötralizasyon testi sonunda elde edilen titre degeri 1/1280 olarak saptanmistir. Plasma and End Product Tests: The virus made following the concentration and purification of the plasma collected as 75 L. The titer value obtained at the end of the neutralization test was determined as 1/1280.
Arjantin°de yapilan bir çalismada SARS-COV-Z virusuna ait spike proteinin reseptör baglama bölgesi rekombinant uygulamalarla çogaltilmis ve hücrelerden elde edilen proteinler affinite kromatografi yöntemi ile saflastirilarak at immunizasyonlarinda kullanilmistir. Elde Edilen sonuçlar atlarda 1210240 antikor titresine kadar ulasildigini ve takip eden plazma saflastirma islemleri sonunda da bu titrenin yüksek oranda korundugunu, dolayisiyla elde edilen son ürünün konvelesan plazmadan yaklasik 50 kat daha fazla titrede nötralizan antikor içerdigi belirtilmistir. Canli SARS-COV kullanilarak yapilan bir çalismada atlarda ilk immunizasyonu takiben 7. haftada 1114210 serum antikor titresine kadar ulasildigi belirtilmistir. Saflastirrna islemini takiben bu titre 1/5120 olarak belirlenmistir. In a study conducted in Argentina, the spike protein of the SARS-COV-Z virus The binding site was amplified by recombinant treatments and obtained from cells. Proteins are purified by affinity chromatography method and used in horse immunizations. used. The obtained results showed that up to 1210240 antibody titers were reached in horses and At the end of the following plasma purification processes, this titer was preserved at a high rate, Therefore, the final product obtained has a titer of approximately 50 times higher than that of convalescent plasma. It is stated that it contains neutralizing antibodies. In a study using live SARS-COV The serum antibody titer of 1114210 was reached in the 7th week following the first immunization in horses. specified. Following purification, this titer was determined as 1/5120.
Bu bulusta gerçeklestirilen inaktif SARS-COV-Z virusu ile yapilan immunizasyon çalismalari sonunda atlardan elde edilen titre degerleri 1/640-1/5120 arasinda bireysel düzeyde degisiklik göstermis olupI satlastirmayi takiben elde edilen ürüne ait nötralizan antikor titre degeri 111280 olarak tespit edilmistir. FAO tarafindan konvelesan plazma titre düzeyi 1/160, bunu temin edilemedigi durumlarda ise 1/80 olarak kabul edilmektedir. Immunization with inactive SARS-COV-Z virus performed in this invention At the end of the studies, the titer values obtained from the horses were between 1/640-1/5120. The neutralizing agent belonging to the product obtained following the trade antibody titer value was determined as 111280. Convalescent plasma titer by FAO level of 1/160 is accepted as 1/80 in cases where this cannot be achieved.
Ebolaya karsi benzer bir çalismada yine VLP kullanilmis ancak çalismada hiperimmun eldesi için rodentler kullanilmistir. Bununla beraber fareler ve kobaylarda yapilan maruz birakma (Challange) testleri sonunda terapötik uygulamanin zamanlamasinin kritik bir önem tasidigi Virus nötralizasyonuna ek olarak FC-bagimli antikor mekanizmalarinin da bu süreçte önemli bir role sahip olabilecegi vurgulanmistir. In a similar study against Ebola, VLP was used again, but in the study, hyperimmune Rodents were used to obtain it. However, exposure in mice and guinea pigs Timing of therapeutic administration at the end of challenge tests is of critical importance. In addition to the virus neutralization it carries, FC-dependent antibody mechanisms are also involved in this process. highlighted that it can play an important role.
Son Ürün Sterilite ve Ekstra Ajan Kontrolleri Sterilite ve ekstra ajan kontrollerinde testlerinde elde edilen son ürünün bakteriyel ve ekstra ajanlar yönünden ari oldugu belirlenmistir. Final Product Sterility and Extra Agent Controls In the sterility and extra agent controls, the bacterial and extra-bacterial It was determined that it was free from agents.
Safsizlik Örnekler Stoktan l/l, l/5 ve l/lO seyreltme ile kuyucuklara yüklenmistir. Analiz indirgen ve indirgen olmayan sartlarda gerçeklestirilmistir. (Sekil 1). SDS-PAGE analizinde yüksek miktar örnek yüklenerek safsizliklarin daha belirgin tespit edilmesi amaçlanmistir. impurity Samples were loaded into the wells at 1/1, 1/5 and 1/10 dilutions from Stock. Analysis reducing and under non-reducing conditions. (Figure 1). high in SDS-PAGE analysis It is aimed to determine the impurities more clearly by loading the sample.
MALDI-TOF Analizi Sonucu: Antiserum içeriginde baskin bilesenin immunglobulin kaynakli F(ab)2 oldugu tespit edilmistir. Örnek içerisinde farkli proteinlerin tespiti için dogrudan kütle analizi MALDl- TOF analizi gerçeklestirilmistir (Sekil 2). Analizde görülen pikler protein agirliklarini göstermektedir. 2, 48 kDa Fe ya da IgG agir zinciri olarak düsünülmektedir. Diger pikler at serumundan gelen kontamine proteinler olabilir seklinde degerlendirilmistir. Analiz sonucunda antiserum örneginin içerisindeki baskin bilesenin immünglobulin kaynakli F(ab)2 oldugu tespit edilmistir. Proteomik analizler ve SDS-PAGE analizi bu bulguyu desteklemektedir. MALDI-TOF Analysis Result: It was determined that the dominant component in the content of the antiserum was F(ab)2 originating from immunoglobulin. has been made. Direct mass analysis MALDl- for the detection of different proteins in the sample TOF analysis was performed (Figure 2). The peaks seen in the analysis indicate the protein weights. shows. As a 2.48 kDa Fe or IgG heavy chain is being considered. Other peaks may be contaminated proteins from horse serum. has been evaluated. As a result of the analysis, the dominant component in the antiserum sample Immunoglobulin-derived F(ab)2 was detected. Proteomic analyzes and SDS-PAGE analysis supports this finding.
SARS-COV-2 virus antiserumu ile indigeyici ve indirgeyici olmayan kosullarda yapilan jel analizleri ve moleküler elek kromatografisi sonuçlari yapilan kütle analizi sonuçlariyla birlikte yorumlandiginda; bu yaklasimla elden edilen sonuçlar asagidaki sekilde özetlenebilir: immünoglobülinlerden olustugu, 3.Tespit edilen yüzdeler orijinal ayristirilmamis antiserumdan yapilan jel elektroforez analizleri, moleküler elek kromatografisi SOnuçlari ve ayristirilmis proteinler ile yapilan elektroforez sonuçlarinda ayni birbirini teyit eder sekilde görüldügü, 4.NR jellerde (örnegin Sekil 3) yüksek miktarda protein içeren 1:10 seyrelti kuyusunda 47 kDa civarinda görülen bantin IGg agir Zinciri olabilecegi; .Ayr1stirilmis proteinlerle yapilan analizlerde 37-50 kDa araligindaki protein bantlarinin toplam miktarinin % 6 ve 10-20 kDa araligindaki proteinlerin miktarinin da yaklasik % 6 olarak belirlendigi, 6. 50-10 kDa arasindaki protein bantlarinin uygun yöntemlerle, örnegin, kütle spektroskopisi veya affinite kromatografisi ile tanimlanabilecegi anlasilmistir. Gel made with SARS-COV-2 virus antiserum under reducing and non-reducing conditions analyzes and molecular sieve chromatography results with the results of mass analysis when interpreted together; The results obtained with this approach are as follows can be summarized: consists of immunoglobulins, 3. Percentage detected by gel electrophoresis from original undifferentiated antiserum analysis, molecular sieve chromatography results and isolated proteins. It was seen in the electrophoresis results in the same way that they confirm each other, 4. 47 in 1:10 dilution wells containing high protein in NR gels (eg Figure 3) The band seen around kDa may be IGg heavy Chain; In the analyzes made with .Ayr1stirilmis proteins, protein bands in the range of 37-50 kDa were found. 6% of the total amount and approximately 6% of the amount of proteins in the 10-20 kDa range determined as, 6. Protein bands between 50-10 kDa by suitable methods, eg mass spectroscopy or by affinity chromatography.
Osmalarite: Avrupa Farmakopesi 2.2.35 maddesine göre yapilan analizlere göre osmolarite degeri 324 mOsm/Kg H20 olarak belirlenmistir. Osmalarity: The osmolarity value is 324 according to the analyzes made according to the European Pharmacopoeia article 2.2.35. It is determined as mOsm/Kg H2O.
Protein tayini: Dumas metodu ile %13 Azot belirlenmistir. Protein determination: 13% Nitrogen was determined by the Dumas method.
Zararsizlik ve pirojenite testleri: Zararsizlik testlerinde baslangiç günü sifirinci gün olarak kabul edilmistir. Bunun ardinda Birinci, üçüncü, besinci ve yedinci günlerde fareler tartilmis ve agirlik artislari kontrol grubuna göre degerlendirilmistir. Ürün üzerinde bir hafta yapilan tartimlarda deneme yapilan gruplarin agirlik artislarinin kontrol grubundan farkli olmadigi görülmüstür. Bulus kapsaminda 6 tavsan üstünde yapilan denemede Tablo l°de belirlenen isi degisimleri kaydedilmis ve toplamda 1,79°C sicaklik artisi belirlenmistir. Saf antiserum numunesinin pirojen etkiye sahip olmadigi anlasilmistir. Harmlessness and pyrogenicity tests: In the harmlessness tests, the starting day was accepted as the zeroth day. behind this On the first, third, fifth and seventh days, the rats were weighed and their weight gains controlled. evaluated by group. In the weighings made for a week on the product, the trial was carried out. it was seen that the weight gains of the groups were not different from the control group. Meet The heat changes determined in Table 1 in the experiment performed on 6 rabbits within the scope of recorded and a total temperature increase of 1.79°C was determined. of pure antiserum sample It is understood that it does not have a pyrogen effect.
Tablo 1. Pirojenite testi sonuçlari. Table 1. Pyrogenicity test results.
Pirojenite Testi Vücut Isi Degisimi °C Tavsan (0:31 Tavsan No Bu bulusta ETLKVET3 (MW306668J susu kullanilmistir. Immunizasyon 4 asamada yapilmistir. Haftada bir olmak üzere dört enjeksiyon yapilmistir. Bir hafta ara ile ilk iki enjeksiyon 1 ml Complete Freund's adjuvan (Sigma F5881) ve 1 ml inaktif antijen; sonraki iki haftaya ait enjeksiyonlar ise 2 ml Incomplete Freund's adjuvan (Sigma F5506i ve 2 ml inaktif antijen içerecek sekilde uygulanmistir. Pyrogenicity Test Body Temperature Change °C Rabbit (0:31 Rabbit No. In this invention, strain ETLKVET3 (MW306668J was used. Immunization in 4 stages has been made. Four injections were given once a week. First two weeks apart injection 1 ml of Complete Freund's adjuvant (Sigma F5881) and 1 ml of inactive antigen; next Two-week injections are 2 ml Incomplete Freund's adjuvant (Sigma F5506i and 2 ml It was applied as containing inactive antigen.
Tavsanlar için her inaktivasyon yöntemine ait 6 tavsan kullanilmistir. Atlarda ise BEI+FA inaktivasyon denemesi için 4”er, FA ve isil inaktivasyon için ise 2'ser at kullanilmistir. For rabbits, 6 rabbits belonging to each inactivation method were used. In horses BEI+FA 4 horses were used for the inactivation trial, and 2 horses each for FA and thermal inactivation.
Enjeksiyonlari takiben gelisen antikor yanitin takibi amaciyla ve (atlar için) 4., 6., 8. haftalarda kan alma ve plazma toplama islemini gerçeklestirilmistir ve sonunda tüm plazmalar havuz yapilarak saflastirma islemine tabi tutulmustur. To monitor the antibody response following injections and (for horses) 4th, 6th, 8th. In weeks, blood collection and plasma collection were performed and finally all The plasmas were pooled and subjected to purification.
Bu bulus sayesinde COV I D-19 tedavisinde ve hastaliktan korunmasinda kullanilacak ve kisa sürede fazla miktarda üretime uygun olan antiserum elde edilmistir. Bulusa konu olan anti serum hastalik belirtileri basladigi zaman verildiginde, vücudun üretmesi gereken savunma hücreleri bir paket olarak verilmis olmaktadir. Bu sayede vücudun yorulmasina gerek kalmadan Covid 19791 karsi Vücudun savunma sisteminin daha hizli cevap vermesi saglanarak, ilaç, yatar hasta maliyeti, is gücü kaybi azalmis olacak, ekonomik gelir saglanacak ve hasta degilken bile belirlenecek dozlar ile kullanildiginda vücutta hazir antikor bulunmus olacaktir. Bu avantaji sayesinde asi ile saglanmasi planlanan korumadan daha fazla koruma daha düsük maliyet ile saglanmis olacaktir. Bulusa konu ürün asi gibi etkinlik gösterecek, belli dozlarda koruyucu olarak risk grubunda bulunan insanlarin özellikle saglik personellerinin kullanmasi için önerilebilecektir. Thanks to this invention, it will be used in the treatment of COV I D-19 and in the prevention of the disease and for a short time. Antiserum suitable for large amount of production was obtained in a short period of time. The subject of the invention is the anti When the serum is given when the symptoms of the disease begin, the defense that the body must produce cells are given as a package. In this way, the body does not need to be tired. Faster response of the body's defense system against Covid 19791 By providing medicine, inpatient costs, loss of labor will be reduced, economic income will be reduced. ready antibody in the body when used with doses to be provided and determined even when the patient is not sick will have been found. Thanks to this advantage, it is more than the protection planned to be provided with the vaccine. more protection will be provided at a lower cost. Activity such as product subject to the invention It will show that people who are in the risk group as a preventative at certain doses, especially health may be recommended for use by staff.
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