Making of Versavo^®

Development

Our approach to biosimilarity

The World Health Organization (WHO) defines the essence of biosimilarity to be primarily ‘the absence of a relevant difference in the parameter of interest’. The WHO guidelines on Biosimilars, or Similar Biotherapeutic Products, describe them as products which are similar in terms of quality, safety and efficacy to an already licensed Reference Biotherapeutic Product ^[1].

A biosimilar is thus similar to its Reference Product, with respect to structural and functional similarity and also clinical safety and efficacy.

Dr. Reddy’s approach to biosimilarity development conforms to the core principles outlined by the WHO guidelines and regulatory agencies worldwide^[2] i.e., design and development based on a comprehensive understanding of the clinically relevant attributes of the Reference Product, and demonstrate evidence for similarity with a stepwise approach (Figure 1), sequentially eliminating any uncertainties about potential differences in the efficacy, safety and patient outcome.

Figure 1: Biosimilarity - A stepwise approach to build totality of evidence
Source: Expectations for Biosimilars, Steven Kozlowski, PDA Biosimilars Conference, Baltimore, 2016

Versavo^® Development History

It is known that biopharmaceuticals produced from a biological systems may exhibit batch to batch variations in product quality attributes^[5]. These variations in attributes are regulated to lie within an acceptable range where existing evidence would predict no adverse impact on safety or efficacy of the product (ICH Q5E). Hence development of Versavo^® has been guided by a thorough understanding of the critical quality attributes and their acceptable ranges obtained by extensive characterization of the Reference Products (RP) licensed in US and EU. It also involves establishing the structure-function relationships, and consolidation of scientific knowledge available from published information / literature on Bevacizumab.

Versavo^®: Proof of biosimilarity

Biosimilarity of Versavo^® was demonstrated based on a stepwise approach, building the foundation first with an extensive structural and functional characterization of both the proposed biosimilar product and the Reference Products. This was followed by evidence from pre-clinical and clinical studies to further establish the similarity to the Reference Products in terms of safety, efficacy and immunogenicity.

Structural and functional similarity of Versavo^® to the Reference Products was demonstrated using an array of advanced analytical methods, which deliver a detailed picture of the physical, chemical and biological characteristics of both the products (Figure 2).

Figure 2: Approach towards establishing the structural-functional similarity
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
C1q= compliment component

Establishing structural similarity

An extensive array of advanced analytical physicochemical methods have been used to compare the protein at various levels of structural hierarchy. The results are summarized in Table 1, with data from a subset of key tests described in (Figure 3).

Table 1: Summary of results of assays to demonstrate structural similarity between Versavo^® and the Reference Product.
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
pI = Iso-electric point, IEX-HPLC = Ion-Exchange High-Performance Liquid Chromatography, SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, HPLC = High-Performance Liquid Chromatography, AUC = Analytical Ultra-Centrifugation, SEC-MALLS = Size Exclusion Chromatography with Multiangle Laser Light Scattering,

Figure 3 (A, B): Orthogonal physicochemical methods demonstrate Structural similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
LC-MS = Liquid Chromatography–Mass Spectrometry

Primary structure (Figures 3A and 3B) : Indicates identical amino acid sequence, and is considered key determinant of all other molecular properties, including functional activity and stability hence impacting clinical safety and/or efficacy. This is verified first at the DNA level using the nucleotide sequencing and then demonstrated at protein level by a peptide map covering the complete sequence and also at positions of key residues for carbohydrate attachments using a combination of chromatographic and mass spectrometric techniques (LC-MS).

Figure 3C: Orthogonal physicochemical methods demonstrate Structural similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
UV CD = Ultraviolet Circular Dichroism

Secondary Structure (Figure 3C) : Multiple lots of Versavo^® and Reference Product subjected to CD analysis in the far UV region showed superimposable spectra indicating that the products have highly similar secondary structures.

Figure 3D: Orthogonal physicochemical methods demonstrate Structural similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished

Tertiary Structure (Figure 3D) : Variations in protein intrinsic fluorescence in the 300-450 nm range, is widely used as a diagnostic criteria for perturbations in the local conformations and global tertiary structures. Data with Versavo^® and Reference Product show superimposable fluorescence spectra, indicating similar tertiary structures.

Figure 3E: Orthogonal physicochemical methods demonstrate Structural similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished

Thermal Stability (Figure 3E) : Differential Scanning Calorimetry is used for determining thermal stability of proteins. Data demonstrates Versavo^® is highly similar to the Reference Product for thermal stability, and indicating similar protein folding and higher order structure based on change in heat capacity.

Conclusions about structural similarity

Results from multiple different assays (Figures 3 A to E and Table 1) demonstrate that Versavo^® is highly similar to the Reference Product with respect to primary, higher order structures and thermal stability – all attributes of clinical significance.

Establishing functional similarity

Once we established that Versavo^® and the Reference Products are structurally similar, we demonstrated functional similarity by biological assays related to binding to target (Fab binding to VEGF) and secondary mediators (Fc binding to mediators in the immune system). The results of functional similarity tests are summarized in Table 2.

Table 2: Summary of results of assays to demonstrate functional similarity between Versavo^® and the Reference Product.
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
HUVEC= Human Umbilical Vein Endothelial Cell, ELISA= Enzyme-linked Immune Sorbent Assay, SPR= Surface Plasmon Resonance, VEGF = Vascular Endothelial Growth Factor, FcRn= neonatal Fc receptor

Figure 4 (A and B): Functional similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished
VEGF = Vascular Endothelial Growth Factor

Biological Activity (Figure 4A and 4B): Functional similarity assessment included (A) potency as measured by inhibition of VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) and (B) binding to the target VEGF protein.

The activity of Versavo^® and Reference Product was highly similar in assays measuring these key activities related to mechanism of action.

Figure 4C: Functional similarity of Versavo^® with Reference Product
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished

Binding to FcRn (Figure 4C): The neonatal Fc receptor (FcRn) binds to IgG in the Fc region and mediates IgG homeostasis in humans and the results demonstrate that Versavo^® and Reference Product has similar binding for FcRn.

Summary of functional characterization

Based on the results from assays related to binding to the target and immune mediators, and the activities leading to the Mechanisms of Action, Versavo^® is demonstrated to be functionally similar to the Reference Product.

Establishing preclinical equivalence

Pharmacokinetic profile analysis

Comparative pharmacokinetic studies of Versavo^® and Reference Product was conducted by administering the intravenous dose to normal male and female Swiss albino mice. The result (Figure 5A and 5B) of overlapping time versus concentration curves shows similar systemic exposure, half-life and clearance, indicating that Versavo^® and Reference Product have similar pharmacokinetic profiles ^[2].

Figure 5 (A, B): Preclinical Pharmacokinetics Profiles of Versavo^® and Reference Product in Swiss Albino Mice [Male (A) and Female (B)]
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished

Establishing Clinical equivalence

The foundation of development of biosimilars is physicochemical and analytical similarity demonstrated with the Reference Product that has extensive clinical data, and many years of established clinical use – and hence an established safety, efficacy profile and dosing regimen(s).

Clinical development of biosimilars, seeks to eliminate any residual uncertainty after structural and functional similarity establishment and to validate that those are not clinically relevant.

Clinical similarity is confirmed by addressing the pillars of clinical similarity assessment, with respect to the Reference Product:

1. Similar PK profile – to be established in the most sensitive, cleanest and most homogeneous population, with no confounding factors: hence in healthy subjects (wherever toxicity profile does not preclude administration to healthy subjects)
2. Similar safety and efficacy profile - comparative efficacy and safety to be assessed in patient population
3. No higher risk of immunogenicity - comparative immunogenicity to be assessed in both healthy subjects and in patients

Clinical studies in healthy volunteers and patients designed and conducted to eliminate bias, enhance sensitivity and ensure certainty^[4].

i. Normal Healthy volunteer (NHV) study^[6] –

This study conducted in 150 healthy volunteers established the three- way pharmacokinetic (PK) equivalence between DRL-Bevacizumab, the EU Reference Medicinal Product and the US Reference Product (Figure 6). Further, similar safety and immunogenicity profiles were observed for the three products.

Figure 6: Mean (±SD) serum concentration–time profiles for all treatments on linear scale (pharmacokinetic population).
Source: Wynne C1, Schwabe C2, Batra SS3, Lopez-Lazaro L4, Kankanwadi S4. Br. J Clin Pharmacol 2018 Oct:84(10):2352-2364. Epub 2018 Jul 26 doi:10.1111/bcp.13691.

The results of this NHV study has been published in the British Journal of Clinical Pharmacology (BJCP).

“A comparative pharmacokinetic study of DRL_Bevacizimab, a candidate biosimilar of bevacizumab, with Avastin^® (EU and US) in healthy male subjects. Wynne C1, Schwabe C2, Batra SS3, Lopez-Lazaro L4, Kankanwadi S4”.

Br J Clin Pharmacol 2018 Oct; 84(10):2352-2364. Epub 2018 Jul 26. doi: 10.1111/bcp.13691.

ii. Patient study in 2 indications - metastatic colorectal cancer (mCRC) & non-small cell lung cancer (NSCLC) –

Well designed and well conducted randomised, double-blinded, multi-centre clinical study, across various centres.

Various salient features of the study include:

• Randomized, controlled, double-blind design to ensure no reporting/ observer bias
• Conducted in the 2 common indications of bevacizumab – metastatic colorectal cancer (mCRC) and Non-small cell Lung Cancer (NSCLC).
  Globally, in both the sexes combined, lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death, while colorectal cancer is the fourth commonly diagnosed cancer and second leading cause of cancer death in the world^[7].
  
• Efficacy: based upon progression free survival (PFS) as the primary end point of the study, with radiological scans read by an independent reviewer(s) to ensure consistency and to eliminate subjective bias
• Comprehensive evaluation of Immunogenicity for both binding and neutralizing antibodies, with assessments both pre-treatment and at periodic intervals during treatment. Analyses performed using validated methods and assays at one central laboratory to ensure high quality of data and reduced variability of results
• Data and Safety Monitoring Board (DSMB) oversight throughout the course of the study (especially focusing on safety aspect of the study)
• Compliant as per the Drugs Controller General of India (DCGI) requirements and the revised Biosimilars Guidelines, India dated 15th August 2016, - more than 100 patients in the test arm (i.e. DRL-Bevacizumab).

Versavo, thus meets all clinical parameters of similarity assessment vs Reference Product (Figure 1).

Figure 7: Clinical parameters of similarity

Versavo^® is Dr. Reddy’s brand of biosimilar bevacizumab. Versavo^® was registered in India in 2019. We have referred to Avastin^® (Bevacizumab, EU and US) as the ‘Reference Products’.

Manufacturing & Quality

Manufacturing Process

Versavo^® was developed through a genetic engineering process that introduces a gene coding for the bevacizumab antibody into Chinese Hamster Ovary cells (CHO) [identical to the Reference Product host cell line]^[1].

The recombinant CHO cells are grown under specific, well-controlled conditions in a bioreactor, that when fed with the right nutrient medium, allows the recombinant cells to multiply and synthesize the protein, releasing it into the surrounding growth medium. A series of purification and chromatographic steps then purify the protein to procure highly purified bevacizumab monoclonal antibody protein.

Figure 1: The manufacturing process
Source: Dr. Reddy’s Laboratories Ltd (2019) Data on file. Unpublished

The purified protein is then formulated with excipients that help to maintain product stability under the recommended shelf life conditions. The formulated protein product is then filled in vials under aseptic conditions to obtain the final drug product.

Vials are stored at designated temperature conditions for distribution to the hospitals and patients.

Process Control

Dr. Reddy’s strives to ensure high product quality during manufacturing till the time product reaches the patient. Our strategy is to produce desired product quality consistently at scale, based on an in-depth understanding of each unit operation that include validated process controls and testing.

Quality

Our manufacturing facility is approved by Regulatory Authority in India and certified for World Health Organization (WHO)-GMP and International Organization for Standardization (ISO) 9001. It is also approved by other global health authorities^[1]. Our product is manufactured under cGMP conditions.

Versavo^® is Dr. Reddy’s brand of biosimilar bevacizumab. Versavo^® was registered in India in 2019. We have referred to Avastin^® (Bevacizumab, EU and US) as the ‘Reference Products’.