Yashraj Biotechnology Limited C-232 TTC Industrial Area ......cost effective customized antibody discovery and manufacturing services to our esteemed customers. Based on desired application,

YBL NEWSLETTER Volume 7, Issue 1, March 2020

Yashraj Biotechnology Limited C-232 TTC Industrial Area, Pawane MIDC, Navi Mumbai 400705, India Website: www.yashraj.com Email: [email protected] Telephone: +91 22 62166800

INSIDE THIS ISSUE • Recombinant

antibodies • New antigen: Native

Thyroglobulin • New antigen:

Recombinant HIV-1p24 • Articles in 2019-2020 • IFCC Awards • Contact Details

ADVISORY BOARD • Mr. Arvind Bhanushali • Mr. Bharat Dagha • Prof. G. Padmanaban • Dr. Paresh Bhanushali

EDITORIAL BOARD • Dr. Chander P. Puri Chief Editor

MEMBERS • Dr Shweta Bhatt • Dr Anjali Karande • Dr. Gauri Awasthi • Dr. Anuj Kumar Gupta • Dr. Kunal Shukla • Dr. Mukesh M Tripathi • Mrs. Prerana Rathod • Mr. Nikhil Rautela

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As we bring to you this new issue of our newsletter, the world is facing an unprecedented healthcare emergency in the form of a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Covid-19. Almost all the countries in the world are victim of this infectious disease, within a short span of about four months the disease has taken the shape of a global pandemic, and the number of infected individuals as well as those dying of the disease has been increasing exponentially.

It is highly commendable that the scientific community has promptly risen to the challenges of offering early diagnosis, and developing both preventive and curative treatment modalities. Highly specific and sensitive in vitro diagnostic methods have been developed, at least eight multi-centric clinical trials have been started to develop an effective vaccine, and number of new drugs are being developed to treat positive patients.

During these trying times, our research teams at Yashraj Biotechnology Ltd have also taken initiative to develop indigenized cost-effective and rapid Q-PCR-based as well as rapid serological tests for Covid-19 diagnosis, and we hope such initiatives would allow both screening of population as well as diagnosis thereby help in curtailing the spread of the disease.

Simultaneously, our company has ventured into manufacturing of recombinant monoclonal antibodies through a synthetic phage display library. This new initiative would allow us to manufacture larger quantities of antibodies in a shorter time frame and at reduced cost, while the process will retain all relevant functional attributes of the displayed nanobodies.

In addition, our basket of antigens has been further expanded through the development of native Thyroglobulin and recombinant HIV-1 p24. The antigens have been developed using stringent quality control parameters that ensure highest purity along with long-term stability compared to best market standards.

In many cases, native antigens have been complemented by manufacturing recombinant antigens which mimic native antigens and provide comparable reliability in in vitro diagnostic kits including their use as Controls and Immunogen by diagnostic industry. For more details please visit www.yashraj.com

Our scientists have also been avidly contributing research and corporate articles including on harnessing the potential of stem cells for preclinical applications, and achieving excellence in biotechnology through its waste to wellness initiative.

The Editorial Board also thanks the entire staff of our company for making generous financial contribution to the Prime Minister Relief Fund as a token of support to deal with the Covid-19 epidemic.

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Our Company Ventures into Manufacturing of Recombinant Antibodies using Phage Display Technology

YBL has pioneered the manufacturing of monoclonal antibodies using mouse-derived hybridoma technology in India. Amongst the various monoclonal antibodies available are those against cancer antigens and other metabolic markers such as anti-CA15-3, anti-CEA, anti-AFP etc., available at very high affinity and some with match pairs available for various immunodiagnostic assays.

Owing to their growing demand, in relatively large quantities and shorter lead times, alternate routes of antibody production had to be considered, prompting YBL to foray into the recombinant antibody development technology. This in vitro technology for the production of recombinant antibodies (Nanobodies) through a synthetic phage display library, allows use of synthetic genes, easy isotype conversion and most importantly does not require animals for immunization purposes, while retaining all relevant functional attributes of the displayed nanobodies, desirable for in vitro diagnostics as well as therapeutic use.

At YBL, we are in the process of developing M13 phage-based, synthetic type of nanobody phage display library, which would allow discovery and manufacturing of recombinant monoclonal antibodies for desired applications. Nanobodies are among the smallest known antigen-binding antibody fragments, and are derived from the heavy-chain only antibodies that occur naturally in the serum of Camelidae.

Synthetic nanobody phage display library being developed by us would offer quick, efficient and cost effective customized antibody discovery and manufacturing services to our esteemed customers. Based on desired application, we will provide HRP or biotin conjugated Nanobody, as a bi-specific antibody, targeting two different antigens or as a full-length antibody fused with either mouse, rabbit or human Fc region. These recombinant antibodies are expected to have superior solubility, stability, with better epitope recognitions and tissue penetration compared to conventional antibodies due to the inherent properties of chosen nanobody format.

Some of the antibodies we have initiated work on are: human anti-CRP (C-Reactive protein), anti-AFP (alpha-fetoprotein), anti-NGAL (Neutrophil associated gelatinase lipocalin), anti-Calprotectin, anti-Prolactin, human anti-SAA (Serum Amyloid A), anti-SHBG (Sex Hormone Binding Globulin), anti-HE4 (Human Epididymis Protein 4), PSA-ACT (Prostate specific antigen /alpha 1- anti chymotrypsin complex), Canine CRP and Feline SAA.

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New Antigen: Native Thyroglobulin

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Thyroglobulin (Tg) is a homodimeric glycoprotein of 660 kDa produced predominantly by the thyroid gland. It acts as a substrate for the synthesis of thyroxin (T4) and triiodothyronine (T3). Thyroglobulin (Tg) is synthesized by the follicular epithelial cells of the thyroid and secreted from the thyroid gland with the stimulation of TSH and/or thyroid stimulating immunoglobulin’s. Circulating Thyroglobulin has a half-life of 65 hours.

Thyroglobulin is a prognostic marker for Graves’ disease. Thyroglobulin levels in the blood are mainly used as a tumor marker for certain kinds of thyroid cancer (particularly papillary or follicular thyroid cancer).

Antigenic activity of purified Tg protein is determined by ECLIA (Roche) at YBL lab. The ratio of immuno-activity and total protein of Thyroglobulin is almost near to 1.0. Initially, there was an uncertainty regarding the presence of slight traces of Anti-Tg in the sample as it was showing a weak signal for Anti-Tg on competitive platform. Further tests were performed to investigate for the presence of Anti-Tg in the sample. The western blot analysis against Immunoglobulin with very high load of 25µg of Tg exhibited negative results. This result was further supported by the ELISA which showed almost negligible amount of antibody in the sample. The YBL standard was also compared with four different market standards in terms of purity and immuno-activity and was found to be at par with the other market standards.

SDS-PAGE profile shows only the bands of Tg in non-reducing condition. Western blot was performed using anti Tg monoclonal antibody. Lane 1: Molecular weight marker; Lane 2:

Thyroglobulin (YBL)

SDS-PAGE (7%) and Western blot analysis of Thyroglobulin

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Data1: YBL sample Data2: Market standard

By HPLC, purity of YBL Thyroglobulin was compared with well-known market standard.

0

0.5

1

1.5

2

2.5

3

0th day 3rd day 8th day 11th day 15th day

Co

nc.

(m

g/m

l)

Days

Accelerated stability study data

-20°C

37°C

50°C

Stability data confirmed the stability of Tg of 2 year at -20°C (by Q10 accelerated stability).

Stability Study of Purified Thyroglobulin

Analysis of Purity of Thyroglobulin by HPLC

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New Antigen: Recombinant HIV-1 p24

Time course of HIV detection and progression of HIV 1 p24

Capsid protein p24 probably forms the conical core of the virus that encapsulates the genomic RNA-nucleocapsid complex. Early detection is crucial when checking for HIV. HIV-1 p24 antigen appears in the blood as early as 14 days after possible infection. HIV antigen (P24) and HIV antibody testing is used for the diagnosis of HIV infection. Recombinant HIV1P24 antigen (UniProt# P12497) was expressed and purified from “transformed E.coli”. Antigenic activity (Cutoff Index: COI) of purified p24 protein was determined by ECLIA (Roche).

SDS-PAGE profile shows single band of SDS-PAGE in reducing condition. Western blot was performed using anti p24 monoclonal antibody.

SDS-PAGE (15%) and Western blot analysis of HIV-1 p24

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By densitometer (Bio-Rad GS-900), purity of recombinant HIV1 P24 meeting the acceptance criteria i.e. ≥ 95% by SDS-PAGE.

Analysis of Purity of Recombinant HIV-1 p24 by Densitometer (Bio-Rad GS-900)

Cross Immune Electrophoresis (CIE) analysis was done using anti-E.coli polyclonal antibody (DAKO) to check the present of any E.coli contamination. No peak was observed in purified

recombinant HIV-1 p24 sample which confirmed no E.coli contamination in preparation.

CIE gel of Recombinant HIV-1 p24 against DAKO anti-E.coli antibody (B0357)

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Stability Study of Purified HIV-1 p24

Stability confirmed the stability of HIV-1 p24 of 2 years at -20°C (by Q10 accelerated stability).

Real time Stability of HIV-1 p24 Control (Spiked in human serum)

To prepare the control, purified recombinant HIV-1 p24 was spiked in human serum. Activity was determined by ECLIA (Roche). HIV-1 p24 control, spiked in human serum is stable for 3 months at

2-8°C.

Open vial stability study of HIV-1 p24 control (Spiked in human serum)

Product has shown good pattern in open vial stability

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Articles Published during 2019-2020

Sr. No. Author Name Title Name of Journal

1 Dr. Shweta Bhatt and Dr. Gauri Awasthi

Stem Cells- a new era in Therapies and Regenerative Medicines

Cutting Edge Magazine

2 Dr. Gauri Awasthi Vitamin D: The Sunshine Bio-marker and Phylogenetic Analysis of the Evolution of Vitamin D Receptors

The Indian Practitioner

3 Dr. Shweta Bhatt The Stem Cell Euphoria-How far can we ride the wave?

IFCC Newsletter

4 Dr. Gauri Awasthi 20 years of excellence in transforming waste to well-being

Biotech Express Magazine

IFCC INTERNATIONAL AWARDS SPONSORED BY YBL

Our heartiest congratulations to Prof. Nader Rifai, Department of Laboratory Medicine, Boston Children’s Hospital, USA for being selected for “2020 IFCC Distinguished Clinical Chemist Award”, sponsored by Yashraj Biotechnology Ltd. Prof Rifai has been actively pursuing research in identifying new biomarkers particularly for cardiovascular diseases, and has developed several high sensitivity CRP assays which have been cleared by FDA for clinical use. We look forward to working jointly with you Prof Nader Rifai for a common cause of offering sensitive and specific in vitro diagnostics for healthcare.

We also extend our heartiest congratulations to Dr. Sandra Quijano, Department of Microbiology, Faculty of Sciences Pontificia Universidad Javeriana, Bogotá – Colombia for being selected to receive the “2020 IFCC Distinguished Women Scientist Award for Contribution to in Vitro Diagnostics”. This award has been instituted at the initiative of and sponsored by Yashraj Biotechnology Ltd. This award has been specifically instituted to recognize extraordinary contributions made by female professionals to the development or utilization of in vitro diagnostics with emphasis on applications in primary healthcare. Prof Quijano has pioneered the immunophenotypic study of leukemia and lymphomas by immunohistochemistry in samples of bone marrow, peripheral blood and lymph nodes of patients with these diseases and in the study of leukemia by flow cytometry, giving way to the first developments of the immunophenotyping of hematological tumors in Colombia. We look forward to working jointly with you Prof Sandra Quijano for a common cause of offering sensitive and specific in vitro diagnostics for healthcare. The awards will be presented on the occasion of the World Lab Congress to be held in Seoul from 6-10 January 2021.

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CONTACT DETAILS

Come and partner with us at Yashraj Biotechnology Limited in our quest to make diagnostics affordable and easily accessible!

For appointments please contact :

Mr. Bharat T. Dagha, Director, Business Development

[email protected]; [email protected]

ADDRESS YASHRAJ BIOTECHNOLOGY LTD.

C232, TTC Industrial Area, MIDC, Navi Mumbai – 400705, Maharashtra, India. Phone No.: +91-22-6216 6800

www.yashraj.com