Hasil Pencarian  ::  Simpan CSV :: Kembali

"Latar BelakangBiosimilar merupakan produk bioterapeutik yang memiliki kemiripan/kesetaraan mutu, dengan originator, sedangkan biobetter merupakan versi produk biologis lain yang dimodifikasi. Tocilizumab (TCZ) merupakan rekombinan antibodi monoklonal manusia yang dapat menghambat interaksi IL6R dengan IL6 saat inflamasi kronis seperti pada penyakit Rhematoid Arthritis (RA). Saat ini, paten TCZ telah habis, oleh karena itu penelitian ini akan dikembangkan kandidat biosimilar dan biobetter TCZ.MetodePenelitian ini merupakan studi in silico dan in vitro untuk pengembangan kandidat biosimilar dan biobetter TCZ dengan mendesain gen kandidat biosimilar TCZ yang dapat terekspresi di sel mamalia dan dilakukan pengujian hasil transfeksi dengan immunofluorescence, pemeriksaan ELISA, SDS-PAGE dan SPR.HasilHasil penelitian didapatkan struktur 3D kandidat biosimilar TCZ nilai Ramachandran Plot 97.16%, score molecular docking biosimilar TCZ light chain dengan IL6R sebesar -16.0 kcal mol-1 dan lebih besar dari nilai kontrol yaitu -12.5 kcal mol-1. Didapatkan nilai RMSF antara IL6R dengan kandidat biosimilar TCZ rerata <2. Hasil transfeksi kandidatbiosimilar TCZ yang dinilai menggunakan ratio of fluorescence intensity memiliki nilai intensitas diatas 2 sedangkan kontrol memiliki nilai 0. Terdapat dua pita pada 50kDa dan 25 kDa sesuai dengan ukuran protein TCZ reference. Koofisien afinitas IL6R dengan kandidat biosimilar TCZ 9.44e-8 dan menyerupai dengan koofisien afinitas IL6R dengan TCZ Actemra® yaitu sebesar 2.64e-8. Dilakukan modifikasi asam amino pada TCZ light chain paten 1 untuk membuat kandidat biobetter yaitu Ala43, Tyr87 dan Gly41. Hasil validasi 3D kandidat biobetter TCZ dengan hasil 96.70% pada Ramachandran Plot, energi bebas TCZ light chain biobetter sebesar -18.7 kcal mol-1.KesimpulanProduk kandidat biosimilar TCZ memiliki ukuran molekul protein yang sesuai dengan originator TCZ (Actemra®). Selain itu produk biosimilar ini terbukti dapat berikatan spesifik dengan IL6R alfa dengan koofisien afinitas menyerupai Actemra® secara in vitro. Desain in silico kandidat biobetter TCZ dengan modifikasi asam amino Ala43, Tyr87 dan Gly41 menunjukkan afinitas ikatan IL6R alfa lebih kuat dibandingkan Actemra® sehingga diharapkan dapat meningkatkan potensi dalam mengatasi badai sitokin.

---

Background

A biosimilar is a biotherapeutic product that closely matches the quality of the original product, while a biobetter is a modified version of another biological product. Tocilizumab (TCZ) is a synthetic antibody derived from human cells that can block the binding of IL6R to IL6, hence reducing chronic inflammation in conditions like rheumatoid arthritis (RA). At now, the patent for TCZ has lapsed, thus this study aims to create biosimilar candidates and improved versions of TCZ, known as TCZ biobetter candidates.

Method

This research involves both in silico and in vitro methods to produce biosimilar and biobetter TCZ candidates. The aim is to build a biosimilar candidate TCZ gene that can be expressed in mammalian cells. The transfection results will be tested using immunofluorescence, ELISA, SDS-PAGE, and SPR investigations.

Results

The research results showed that the TCZ biosimilar candidate possessed a Ramachandran Plot value of 97.16% for its 3D structure. Additionally, the molecular docking score of the TCZ light chain biosimilar with IL6R was -16.0 kcal mol-1, which was higher than the control value of - 12.5 kcal mol-1. The analysis showed that the average Root Mean Square Fluctuation (RMSF) value between IL6R and TCZ of the biosimilar candidate was less than 2. The TCZ transfection results of the biosimilar candidate were evaluated by measuring the ratio of fluorescence intensity. The biosimilar candidate had an intensity value more than 2, while the control had a value of 0. Two bands were observed at 50 kilodaltons (kDa) and 25 kDa, corresponding to the size of the reference TCZ protein. The IL6R signal affinity of the biosimilar candidate TCZ is 9.44e-8, which is comparable to the IL6R signal affinity of Actemra® TCZ, which is 2.64e-8. The TCZ light chain patent 1 underwent amino acid changes to generate improved biobetter candidates, specifically Ala43, Tyr87, and Gly41. The 3D validation results of TCZ biobetter show a yield of 96.70% on the Ramachandran Plot. Additionally, the free energy of TCZ light chain biobetter is -18.7 kcal mol-1.

Conclusion

The TCZ biosimilar candidate product has a protein molecular size that is identical to the TCZ (Actemra®). Furthermore, this biosimilar product has demonstrated the ability to selectively attach to IL6R alfa with a signal affinity comparable to Actemra® in laboratory tests. The computational design of the biobetter TCZ candidate, incorporating alterations to the amino acids Ala43, Tyr87, and Gly41, demonstrates an enhanced affinity for IL6R alfa compared to Actemra®. This raises the expectation that it may enhance its efficacy in mitigating cytokine storms."

"Latar Belakang: Avian influenza merupakan penyakit infeksius yang mudah menular secara aerosol. Salah satu subtipenya yakni H9N2 mewabah pertama kali di wilayah peternakan unggas di Indonesia pada tahun 2016 dan dilaporkan membawa gen yang cenderung menginfeksi sel mamalia. Antiviral berbasis sialidase dikembangkan sebagai pencegahan dan pengobatan infeksi virus saluran pernafasan seperti avian influenza yang membutuhkan sialic acid sebagai pintu masuknya virus ke dalam sel. Penelitian ini bertujuan mengembangkan antiviral berbasis sialidase asal bakteri Pasteurella multocida, khususnya terhadap infeksi subtipe H9N2.Metode: Penelitian diawali dengan kultur Pasteurella multocida yang secara genetik hanya mengandung satu jenis sialidase yakni NanB. Sialidase dipurifikasi melalui metode kloroform, dan dilanjutkan dengan ion exchange chromatography dan affinity chromatography. NanB sialidase yang dihasilkan diuji aktivitasnya pada suhu, pH dan lama waktu inkubasi yang berbeda. Uji toksisitas dan spesifisitas NanB sialidase dilakukan dengan menggunakan berbagai dosis sialidase pada media sel darah merah ayam dan kelinci serta sel MDCK. Dilanjutkan dengan uji hambatan infeksi virus avian influenza H9N2 dan ekspresi gen penyandi apoptosis sebagai respon sel terhadap infeksi avian influenza H9N2.Hasil: Pasteurella multocida B018 terbukti menghasilkan NanB sialidase dengan berat molekul sekitar 55 kDa. NanB sialidase yang dihasilkan bersifat stabil pada suhu 37℃ dan pH 5 hingga 7 meskipun mengalami penurunan aktivitas pada 72 jam inkubasi. Dosis 0.258 U/ml merupakan dosis toksik yang melisiskan sel darah merah dan merusak sel MDCK. Dosis efektif yang mampu menghirolisis sialic acid adalah 0.129 U/ml, namun NanB sialidase cenderung menghidrolisis sialic acid Neu5Acα(2,6)-Gal pada mamalia. Dosis tersebut juga mampu menghambat ikatan virus H9N2 pada sel darah merah serta menekan jumlah infeksi virus pada sel MDCK tanpa menyebabkan peningkatan ekspresi gen penyandi apoptosis sel. Kesimpulan: NanB Sialidase asal bakteri Pasteurella multocida berpotensi sebagai antivirus avian influenza melalui hilangnya sialic acid akibat proses hidrolisis yang terjadi.

---

Background: Avian influenza is an infectious disease easily transmitted by aerosol. One of the subtypes, H9N2, first appeared in poultry farming areas in Indonesia in 2016 and was reported to carry a gene that tends to infect mammalian cells. Sialidase-based antivirals were developed to prevent and treat viral respiratory infections such as avian influenza, which require sialic acid as the entry point for viruses to enter cells. This study aims to develop a sialidase-based antiviral from the Pasteurella multocida, especially against infection with the H9N2 subtype.

Methods: The research started with Pasteurella multocida culture, which contains only one type of sialidase, NanB. Sialidase was purified by the chloroform method and followed by ion-exchange chromatography and affinity chromatography. NanB sialidase was tested for its activity at different temperatures, pH, and incubation times. The toxicity and specificity test of NanB sialidase was carried out using various doses of sialidase on chicken and rabbit red blood cells and MDCK cells, followed by the inhibition test of avian influenza H9N2 virus infection and expression of apoptotic coding genes as a cell response to avian influenza H9N2 infection.

Results: Pasteurella multocida B018 produced NanB sialidase with a molecular weight of about 55 kDa. The resulting NanB sialidase was stable at 37℃ and pH 5 to 7, although it decreased in activity at 72 hours of incubation. The dose of 0.258 U/ml is a toxic dose that lyses red blood cells and destroys MDCK cells. The effective dose capable of hydrolyzing sialic acid is 0.129 U/ml, but NanB sialidase tends to hydrolyze Neu5Acα(2,6)-Gal sialic acid in mammals. This dose also inhibited the binding of the H9N2 virus on red blood cells. It suppressed the number of viral infections in MDCK cells without increasing the expression of genes encoding cell apoptosis.

Conclusion: NanB Sialidase from Pasteurella multocida has the potential as an antiviral for avian influenza through the loss of sialic acid due to the hydrolysis process"