BIOSIMILARS

Definition

Reference Product vs. Biosimilar Product

Why Biosimilars Are Complex (Unlike Generic Drugs)

• Large molecules (4,000 to >140,000 Daltons vs. <500-900 Da for small molecules)
• Structurally complex - with primary, secondary, and tertiary/quaternary protein structures, plus post-translational modifications (glycosylation, sialylation)
• Process-dependent - even minor changes in the manufacturing process can alter the product
• Immunogenic - can act as antigens and activate the immune system

Demonstrating "Highly Similar" with "No Clinically Meaningful Difference"

1. Physicochemical characterization - structure, quality, purity
2. Functional characterization - biological activity
3. Non-clinical studies - in vitro and in vivo PD studies, toxicity
4. PK/PD analysis - pharmacokinetic and pharmacodynamic comparisons
5. Clinical studies - safety, efficacy, and immunogenicity

Interchangeability

• Produce the same clinical result as the reference product in any given patient
• Show no increased safety risks when alternating/switching between the biosimilar and reference product

Biosimilars vs. Generic Drugs (Key Differences)

Regulatory Aspects

In India

• Drugs and Cosmetics Act, 1940
• New Drugs and Clinical Trials Rules, 2019 (NDCT 19)

1. IBSC (Institutional BioSafety Committee) - initial review
2. RCGM (Review Committee on Genetic Manipulation), DBT - authorizes research and development
3. GEAC (Genetic Engineering Appraisal Committee), MoEF - approves applications involving genetically modified organisms
4. CDSCO (Central Drugs Standard Control Organization) headed by DCGI - approves clinical trials, new drugs, manufacturing, and marketing

By EMA (European Medicines Agency)

By US FDA

Conclusion

DATA REQUIREMENT FOR THE APPROVAL OF SIMILAR BIOLOGICS

1. ANALYTICAL AND QUALITY CHARACTERIZATION DATA

• Physicochemical properties
• Biological activity
• Immunological properties
• Functional assays
• Purity (process- and product-related impurities, etc.)
• Contamination
• Stability
• Strength and content

This is the foundation step - if the biosimilar cannot be shown to be physicochemically and functionally similar to the reference product, further studies are not meaningful.

2. NON-CLINICAL STUDIES

a) In Vitro Studies

• Cell-based bioassays (e.g., cell proliferation assays or receptor binding assays)

b) In Vivo Studies

• PD (Pharmacodynamic/biological activity) studies in specific animal models

c) Toxicity Study

• At least one repeat dose toxicity study in a pharmacologically relevant species, conducted with the intended route of administration
• Other toxicity studies (safety pharmacology, reproductive toxicity, mutagenicity, carcinogenicity) are not required unless warranted by repeat dose toxicity studies

d) Immunogenicity Study

• Results of local tolerance (part of repeat dose or standalone test) should be performed as part of the sub-chronic toxicity study

3. CLINICAL STUDIES

Phase I

• Comparative PK (Pharmacokinetics) and PD (Pharmacodynamics) studies
• PK/PD relationship is evaluated
• PD evaluation may be done as part of Phase III study (usually 1 or 2 phase clinical trials, depending on indications and biology)

Phase III

• Comparative efficacy and safety/immunogenicity studies (usually 1 or 2 clinical trials, depending on number of indications and safety profile of the biologics)
• Equivalence design study is preferred
• Non-inferiority design needs to be justified
• Comparative safety and efficacy clinical trial can be waived if:
  • (I) Comparable quality established by physicochemical and in vitro techniques
  • (II) Comparable in all pre-clinical evaluation
  • (III) Comparable clinical PK-PD including PD markers and immunogenicity data
  • (IV) Post-marketing risk management plan is provided (cannot be waived if one of the above criteria is absent)

Extrapolation to Other Indications

• It may be possible to extrapolate data to other indications if the same mechanism of action (MOA)/receptors is involved, and similar safety, efficacy, preclinical, and quality data exist for reference biologics for that indication

4. REFERENCE PRODUCT REQUIREMENTS

• The reference biologic must be licensed/approved in India or ICH countries and must be the innovator's product
• If the reference biologic is not marketed in India, it should have been licensed in any ICH country (EU, Japan, US, Canada, Switzerland)

5. INTERCHANGEABILITY DATA (Additional Requirements - US FDA)

• The proposed interchangeable product is expected to produce the same clinical result as the reference product in any given patient
• For a product administered more than once, switching between the proposed interchangeable product and the reference product does not increase safety risks or decrease effectiveness compared to using the reference product without switching

• Strength and robustness of comparative analytical studies showing similar structure and function (e.g., very few analytical differences may provide strong support)
• Similarity of PK and PD profiles between the biosimilar and reference product
• Pre-existing safety information about the reference product (e.g., known immune responses may require more rigorous immunogenicity evaluation)

SUMMARY TABLE

ZEBRAFISH (Danio rerio)

Introduction

Scientific Classification

Features of Zebrafish

• Small transparent freshwater fish; standard length ~40 mm
• Body is clear coloured with black stripes running along the body; fusiform shaped
• Caudal fin is striped; dorsal fin is navy-blue coloured
• Being clear coloured, it is an excellent model for fertility and embryonic research
• Omnivorous - feeds on zooplankton, insects, insect larvae, and phytoplankton
• Every stage of growth (zygote, blastula, gastrula, etc.) can be easily appreciated
• Organogenesis and morphogenesis can be easily followed and observed
• The zebrafish genome contains 25 chromosomes, similar to humans
• About 70% of human genes have their analogues in Danio rerio

Unique Characteristics Making Zebrafish an Important Tool for Drug Development

1. High molecular similarity with humans - both exhibit high degree of similarity in molecular mechanisms of development and cellular physiology
2. External fertilization and development - permits direct observation and manipulation of embryos in a wide variety of laboratory conditions; embryos are <1 mm in diameter, distributable into 96- or 384-microtitre well plates; a single embryo can be maintained in 100 mL of embryo medium for days
3. High reproductive output - a pair can produce 100-300 embryos weekly, facilitating statistically significant sample sizes at minimal cost; suitable for medium- and high-throughput chemical screening for preclinical drug discovery and toxicological/teratological evaluation
4. Rapid embryonic development - embryos execute evasive maneuvers within 24 hr post-fertilization (hpf); most major organs (gut, vasculature) are in place by 2 days post-fertilization; embryogenesis is complete by 5 days
5. Optical transparency - embryos and early adults are optically transparent, facilitating direct observation of internal organs by light microscopy
6. Drug permeability - during organogenesis, embryos are permeable to small molecules and drugs, providing easy access for drug administration and vital dye staining
7. Gene expression manipulation - specific proteins can be suppressed ("knock-down") using morpholino antisense oligodeoxynucleotides; embryos can be microinjected with capped mRNA or plasmids; this enables "knock-out/knock-in" approaches widely used in mice

Uses of Zebrafish (Disease Models)

1. Model for Diet-Induced Obesity

• Induced by feeding Artemia nauplii (brine shrimp)
• Shows increased BMI, hepatic steatosis, hypertriglyceridaemia, and dysregulation of lipid metabolism genes
• Also induced by high-cholesterol diet - leads to increased body weight, triglycerides, and lipid deposition in the liver

2. Model for Glucose Metabolism and Type 2 Diabetes Mellitus

• Diabetes induced by immersing zebrafish in high glucose solution for 14 days
• Mutant zebrafish with insulin receptor (a and b gene) knockout given high fat diet leads to hyperglycaemia, visceral adiposity, fatty liver - similar to human lipodystrophy

3. Model for Dyslipidaemia and Atherosclerosis

• Induced by changing from standard diet to high fat diet
• Causes increased cholesterol, triglycerides, and LDL leading to atherosclerosis
• Histopathological changes are very similar to human atherosclerosis

4. Model for Non-alcoholic Fatty Liver Disease (NAFLD) and Other Liver Disorders

• Accumulation of excess fat in the liver leads to steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma
• Zebrafish liver is similar to human liver in cellular structure, function, and genetics
• Immersion in 6% fructose leads to hepatic steatosis

5. Model for Mental Disorders

• A novel chemokine-like gene family, samdori (sam), is involved in mental disorders
• Sam2-knockout animals show autism and anxiety-related disorders
• FAM50A gene is identified as a cause for Armfield X-Linked Intellectual Disability (XLID) syndrome

6. Model for Developmental Disorders

• Transparent body and external egg development allows observation of organ development at each stage
• Example: Potocki-Shaffer syndrome (PSS) - affects formation of bones, nerve cells, and brain; caused by interstitial deletion of band p11.2 in chromosome 1132
• Using phf21a-knockdown zebrafish, developmental disorders and drugs used in PSS can be evaluated

7. Model for Cancer

• High fertility rate, cost-effective genetic manipulation, and high-resolution imaging make it robust for cancer research
• Tumours induced in zebrafish are histologically and molecularly similar to human tumours
• CRISPR/Cas9 technology is used to generate cancer-relevant point mutants in the tp53 gene
• Cancer cells can even be implanted in zebrafish body to observe various stages of tumour development

8. Model for Toxicity Studies

• Established model for drug toxicity studies; determines toxicity in various samples through high-throughput screening
• Zebrafish embryotoxicity model is at the leading edge of toxicology research
• Advantages: short analysis time, transparent embryos, short life cycle, high fertility, and genetic similarity
• Toxicity studies range from assessing toxicity of bioactive compounds to crude extracts from plants

Advantages of Zebrafish

• Smaller size, transparent body, minimal cost in husbandry and breeding
• Large numbers can be kept in a small limited area (only requires an aquatic environment)
• Lay ~200 transparent eggs at a time, which develop outside the female's body - allows observation of organ development at each stage
• Genome is sensitive to manipulations - genetic changes can be induced at each stage of development
• Excellent tool for screening drugs for diseases like Huntington's disease, Alzheimer's disease, sideroblastic anaemia, and ADHD
• Can be used to induce diabetes, obesity, osteoporosis, and cardiovascular diseases (e.g., arrhythmia)
• Cancer cells can be implanted to observe stages of tumour development

Disadvantages of Zebrafish

• Absence of mammalian organs like breast tissue, lungs, and prostate
• Skin does not contain some essential components present in humans
• Adult zebrafish are not suitable for high-throughput screens due to their large size
• Human metabolism process is different - metabolising enzymes (e.g., CYP450s) are not fully characterised
• Microsporidiosis and mycobacteriosis are two infectious diseases common in zebrafish colonies, which can show false results

Conclusion

ZEBRAFISH (Danio rerio)

Introduction

Scientific Classification

Key Features

• Small (~40 mm), transparent body with black stripes
• Omnivorous; external fertilization
• Each pair produces 100-300 embryos/week
• Embryogenesis complete by 5 days post-fertilization
• Optically transparent embryos allow direct observation by light microscopy
• ~70% human gene homology; 25 chromosomes

Why Zebrafish is Used in Drug Development

1. High molecular similarity with humans
2. External development - easy embryo observation and manipulation
3. High reproductive output - suitable for high-throughput screening
4. Rapid embryonic development
5. Optical transparency - internal organ observation without dissection
6. Permeable to small molecules/drugs during organogenesis
7. Gene manipulation possible ("knock-out/knock-in" via CRISPR/Cas9, morpholinos)

Disease Models

Advantages

• Small size, low maintenance cost
• Transparent body - direct organ visualization
• High fertility (~200 eggs at a time)
• Genome easily manipulated
• Useful for screening drugs for Alzheimer's, Huntington's, ADHD, arrhythmia, diabetes

Disadvantages

• No mammalian organs (breast, lungs, prostate)
• CYP450 enzymes not fully characterized
• Adult fish not suitable for high-throughput screens
• Susceptible to microsporidiosis and mycobacteriosis (can give false results)

Conclusion

Allocation Concealment vs. Blinding in Clinical Trials

Core Distinction at a Glance

Allocation Concealment

Definition

Purpose

When it Acts

• Operates before randomization is revealed
• Once a patient is enrolled and assigned, allocation concealment has served its purpose

Methods

• Sequentially numbered, opaque, sealed envelopes (SNOSE)
• Central randomization (phone/web-based, most robust)
• Pharmacy-controlled randomization
• Sequentially numbered drug containers

Key Point

Blinding (Masking)

Definition

Purpose

When it Acts

• Operates after randomization - throughout the conduct and assessment of the trial

Types

Methods

• Placebo - identical-looking dummy treatment
• Double-dummy technique - used when two active treatments have different forms
• Identical packaging of active drug and placebo
• Blinded outcome assessors - separate from treating team

Key Point

How They Work Together

Patient approaches trial
        ↓
[ALLOCATION CONCEALMENT acts here]
→ Enrolling clinician does NOT know
  what treatment next patient will get
        ↓
Patient enrolled and randomized
        ↓
[BLINDING acts from here onwards]
→ Patient, clinician, assessor do NOT know
  which treatment the patient received
        ↓
Outcome assessed and analysed

Bias Each One Prevents

Common Misconceptions

Summary

• Allocation concealment protects the integrity of the randomization process - it prevents bias in who gets enrolled into which group
• Blinding protects the integrity of the trial conduct and outcome assessment - it prevents bias in how participants are treated and outcomes are measured
• Both are needed for a high-quality RCT
• Allocation concealment failure leads to overestimation of treatment effect by up to 40% (Schulz et al., Lancet 1995)
• A trial can have good allocation concealment but poor blinding, and vice versa - they must be independently assessed when evaluating trial quality (e.g., in Cochrane Risk of Bias tool)