This presentation contains recommendations and requirements for the design of bioanalytical testing used in comparibility studies for biosimilar drug development using rituximab as an example

1. Current Practice in Bioanalytical Methods
for Biosimilars:
Case Study of Rituximab Assay Development for
PK, ADA and Nab
Xun Wang, Ph.D.
Department of Translational Medicine
QPS, LLC

2. Top 10 Prescription Drugs in 2016 (*)
Drug Name Innovator Indication Mech. Of WW Sales Billion ($)
(Patent Expiry) Company Action 2009 →2016
Humira™ (2016) Abbott Rhematoid Arthritis TNFαMAb 5.6 → 10.1
Avastin™ (2019) Roche Colorectal Cancer VEGF MAb 5.7 → 8.9
Enbrel ™ (2012) Pfizer, Amgen & Rhematoid Arthritis TNFα Inhibitor 6.5 → 7.3
Takeda
Rituxan™ (2018) Roche Non-Hodgkins Anti-CD20 MAb 5.6 → 6.8
Lymphoma
Crestor ™ (2012) AstraZeneca/ Anti-Hyperlipidemics Small Molecule 4.8 → 6.3
Shionogi/Chiesi Chemistry
Herceptin™(2019) Roche Breast Cancer Her2/neu MAb 4.9 → 6.2
Remicade ™ (2014) JNJ Rhematoid Arthritis TNFα MAb 5.9 → 5.7
Lantus ™ (2018) Sanofi Aventis Diabetics Insulin 4.3 → 5.3
Advair ™ (2011) GSK Bronchodilators Small Molecule 8.0 → 5.2
Chemistry
Prolia ™ (2013) Amgen/Daichii Osteoporosis Bone Calcium 0.0 → 5.2
Sankyo/GSK Reg. MAb
(*) 8 out of the 10 top-selling drugs in the US are biologics. By 2016, some of which will face biosimilar entry.

3. Biosimilar
The biological product is highly similar to the reference product
notwithstanding minor differences in clinically inactive components,” and
that “there are no clinically meaningful differences between the biological
product and the reference product in terms of the safety, purity, and
potency of the product.”
Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act.

4. Biosimilar Industry Today
Market Intelligence
$378M in July, 2010 – June, 2011
Forecast to rise to $2.6B by 2015
Development Cost
$2M – $4M (small molecule generic)
$100M - $200M (large molecule biosimilar)
Key Players: Innovator companies, Emerging market
Major Alliances announced in recent years
Amgen -Watson: Oncology antibody products
Roche - Emcure Pharmaceuticals (India CMO): Herceptin and Mabthera
Merck - Parexel
Biocon (India) - Mylan: Biosimilar monoclonal antibodies
Pfizer - Biocon: Insulin and insulin analogs (The deal was prematurealy terminated in
March)
Momenta (Cambridge, MA) – Baxter International: Enoxparin plus 6 other unspecified
biosimilars
Biogen Idec – Samsung (Korea)

5. FDA Guidance
Public Health Services Act (e.g., erythropoietin)
The Biologics Price Competition and Innovation Act of 2009 (BPCI).
Quality Considerations in Demonstrating Biosimilarity to a Reference Protein
Product (February, 2012).
Scientific Considerations in Demonstrating Biosimilarity to a Reference Product
(February, 2012).
Biosimilars: Questions and Answers Regarding Implementation of the Biologics
Price Competition and Innovation Act of 2009 (February, 2012).

6. Bioanalytical Approaches
PK Assay:
1 Assay vs 2 Assays
Reference standard
QCs
ADA/Nab Assays:
1 Assays vs 2 Assays
Positive controls
Comparable activity and sensitivity

7. QPS Validated Bioanalytical Methods for
Biosimilar
Currently QPS has validated methods for the following biosimilars of market
potential:
Anti-Factor IIa and Anti-Factor Xa assays for Enoxaparin (Activity Assay)
Anti-Factor IIa and Anti-Factor Xa assays for Dalteparin (Activity Assay)
Rituximab (MabtheraTM, RituxanTM): Human PK method (ELISA, Gyrolab)
Rituximab Human ADA methods (MSD ECL)
Rituximab Human Nab methods (Cell based functional)
Bevacizumab (AvastinTM) Human PK method (ELISA)
Darbepoietin Human PK method (ELISA)
Trastuzumab (HerceptinTM) Human PK method (ELISA)
Cetuximab (ErbituxTM) Human PK method (ELISA)
FSH (PuregonTM, Gonal-FTM) (Immulite)

8. Case Study: Rituximab PK, ADA and Nab
Rituximab (Mabthera, Rituxan) is genetically modified monoclonal antibody that
targets CD20 on B cell surface.
Treat solid tumor of lymphoid cells and rheumatoid arthritis with excessive or
dysfunctional B cells.
The mechanisms of action is not fully understood.
Direct signaling
Complement-dependent cytotoxicity
Antibody-directed cell-mediated cytotoxicity

9. Case Study: Rituximab PK
PK Assay Development (ELISA)
2
1 Assay 1.5
1
Reference standard: Innovator
0.5
QCs: Innovator and Biosimilar
0
10 100 1000 10000
Rituximab Concentration (ng/mL)
y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2
Standard Curve (Std: Conc. vs OD) 0.064 1.22 3542.079 3.361 0.999

10. Case Study: Rituximab PK
PK Assay Development (ELISA)*
Validation Items Results
Assay Range 100 - 5000 ng/mL (HUSE)
QC intra/Inter Precision 2.9% to 10.7%
QC intra/Inter Accuracy -8.8% to 9.5%
Matrix Selectivity (low and high QC) At least 85% within ±25% for solid tumor and
RA serum lots
Dilution Linearity 500,000 ng/mL diluted up to
2,000–fold;
ADA Interference At least 1 µg/mL at low QC and
10 µg/mL at high QC
Stability in HUSE -200C/-700C/RT/FT
* Data shown here are from the Rituximab Innovator

11. Case Study: Rituximab PK
PK Assay Development (Gyrolab)
Alexa labeled -
anti human IgG
A high throughput assay platform with Ab
miniaturization and automation Rituximab
Flow
Biotinylated-Rat
Wide assay range: 90 – 60,000 ng/mL in HUSE anti ID rituximab
Validated for Innovator SA analysis
Streptavidin-coated bead
X. Liu et. al. Journal of Immunological Method, 2012

12. Case Study: Rituximab ADA
ADA Assay Development
2 Assays (Innovator and Biosimilar)
Assay format: Bridging with labeled Innovator
or Biosimilar
Positive controls
• Anti-Rituximab Id
• Rabbit anti-Rituximab Polyclonal
• Anti-Biosimilar Id

13. Case Study: Rituximab ADA
ADA Assay Development (Innovator)*
Validation Approaches Results
Items
Screening cut point At least 50 individual ST or 1. Floating cut point
(SCC) RA lots 2. Significantly different SCPs
between matrixes
3. Similar between Innovator and BSI
Confirmatory cut At least 28 individual ST or Similar CCPs between innovator and
point (CCP) RA lots spiked with drug BSI
PC Crossing Check Anti-Biosimilar/Innovator PC showed similar activity in both
Abs in Innovator/BSI assay innovator and BSI assays
Assay sensitivity Determined for all three Sensitivities are similar between
positive control antibodies innovator and BSI assays (< 20 ng/mL)
Drug Tolerance Determined at low and high Low and high PC can tolerate at least
PC (50, 400 ng/mL) 12 and 50 µg/mL Rituximab.
* Data shown here are from the Rituximab Innovator

14. Case Study: Rituximab Nab
Nab Assay Development
2 Assays (Innovator and Biosimilar)
Assay format: Cell based
• Binding?
• Functional -- Go
Positive controls
• Anti-Rituximab Id
• Rabbit anti-Rituximab Polyclonal
• Anti-Biosimilar Id

15. Case Study: Rituximab Nab
Nab Assay Development
Rituximab MOA:
• Complement-dependent cytotoxicity (CDC)
• Antibody-dependent cell-mediated cytotoxicity (ADCC)
• Apoptosis
• A non-radioactive complement-dependent cytotoxicity assay
for anti-CD20 monoclonal antibody (Gazzano-Santoro et. al., Journal
of Immunological Methods, 1997)
http://minimednews.wordpress.com/

16. Case Study: Rituximab Nab
Nab Assay Development
Method Development
Cells growth and density curve: WIL2-S
Rituximab dose killing curve: 70-80% killing
Rabbit complement dose curve
Incubation time
Matrix Evaluation (ST and RA): 1:100
• Specificity
• Selectivity
Compare the biosimilar to the innovator
PC cross check

17. Case Study: Rituximab Nab
Nab Assay Development (Innovator)*
Validation Approaches Results
Items
Screening cut point At least 50 individual ST or 1. Floating cut point
(SCC) RA lots 2. Significantly different SCPs
between matrixes
3. Similar between Innovator and BSI
Immunodepletion At least 28 individual serum Similar between innovator and BSI
lots treated with Protein A/G
PC Crossing Check Anti-Biosimilar/innovator PC showed similar activity in both
Abs in Innovator/BSI assay innovator and BSI assays
Assay sensitivity Determined for all three Currently ongoing (~ 50 µg/mL)
positive control antibodies
Drug Tolerance Determined by High PC Can tolerate 10 µg/mL Rituximab at
titering in the presence of the estimated assay sensitivity
various drug levels.
* Data shown here are from the Rituximab Innovator

18. Conclusion
PK Assay: One assay is sufficient to measure both innovator
and biosimilar drugs.
• Reference Standard: Innovator/BSI
• QCs: Innovator/BSI
Immunogenicity (ADA and Nab) requires two assays, which
are best to develop simultaneously and achieve:
Similar screening and confirmatory cut points
Similar sensitivity
Similar PC cross reactivity
Similar drug tolerance

19. Acknowledgement
Joy He Avery Tolosa Jiannian Zhou
Frank Liu YinLing Li Margaret Ma
Laurelle Calliste Breann Barker
Christina Xia Roni Weaver
Yun Shen
LingSing Chen
Hui Zhang
Holly Shen
Chad Asher