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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

QPS Biosimilar Bioanalytical Approaches

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Page 1: QPS Biosimilar Bioanalytical Approaches

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 MedicineQPS, LLC

Page 2: QPS Biosimilar Bioanalytical Approaches

Top 10 Prescription Drugs in 2016 (*)

(*) 8 out of the 10 top-selling drugs in the US are biologics. By 2016, some of which will face biosimilar entry.

Drug Name(Patent Expiry)

Innovator Company

Indication Mech. Of Action

WW Sales Billion ($)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 & Takeda

Rhematoid Arthritis TNFα Inhibitor 6.5 → 7.3

Rituxan™ (2018) Roche Non-Hodgkins Lymphoma

Anti-CD20 MAb 5.6 → 6.8

Crestor ™ (2012) AstraZeneca/ Shionogi/Chiesi

Anti-Hyperlipidemics Small Molecule Chemistry

4.8 → 6.3

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 Chemistry

8.0 → 5.2

Prolia ™ (2013) Amgen/Daichii Sankyo/GSK

Osteoporosis Bone Calcium Reg. MAb

0.0 → 5.2

Page 3: QPS Biosimilar Bioanalytical Approaches

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.”

Biosimilar

Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act.

Page 4: QPS Biosimilar Bioanalytical Approaches

Biosimilar Industry Today

Market Intelligence$378M in July, 2010 – June, 2011Forecast 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 yearsAmgen -Watson: Oncology antibody productsRoche - Emcure Pharmaceuticals (India CMO): Herceptin and Mabthera Merck - ParexelBiocon (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 biosimilarsBiogen Idec – Samsung (Korea)

Page 5: QPS Biosimilar Bioanalytical Approaches

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).

Page 6: QPS Biosimilar Bioanalytical Approaches

Bioanalytical Approaches

PK Assay:1 Assay vs 2 AssaysReference standardQCs

ADA/Nab Assays:1 Assays vs 2 AssaysPositive controlsComparable activity and sensitivity

Page 7: QPS Biosimilar Bioanalytical Approaches

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)

Page 8: QPS Biosimilar Bioanalytical Approaches

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

Page 9: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab PK 

1 Assay

Reference standard: Innovator

QCs: Innovator and Biosimilar

PK Assay Development (ELISA)

Rituximab Concentration (ng/mL)

10 100 1000 100000

0.5

1

1.5

2

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

Page 10: QPS Biosimilar Bioanalytical Approaches

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

Page 11: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab PK 

PK Assay Development (Gyrolab)

A high throughput assay platform with miniaturization and automation

Wide assay range: 90 – 60,000 ng/mL in HUSE

Validated for Innovator SA analysisStreptavidin-coated bead

Alexa labeled -anti human IgG

Ab

Biotinylated-Rat anti ID rituximab

Rituximab

Flow

X. Liu et. al. Journal of Immunological Method, 2012

Page 12: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab ADA 

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

ADA Assay Development

Page 13: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab ADA 

ADA Assay Development (Innovator)*

Validation Items

Approaches Results

Screening cut point(SCC)

At least 50 individual ST or RA lots

1. Floating cut point2. Significantly different SCPs

between matrixes3. Similar between Innovator and BSI

Confirmatory cut point (CCP)

At least 28 individual ST or RA lots spiked with drug

Similar CCPs between innovator and BSI

PC Crossing Check Anti-Biosimilar/Innovator Abs in Innovator/BSI assay

PC showed similar activity in both innovator and BSI assays

Assay sensitivity Determined for all three positive control antibodies

Sensitivities are similar between innovator and BSI assays (< 20 ng/mL)

Drug Tolerance Determined at low and high PC (50, 400 ng/mL)

Low and high PC can tolerate at least 12 and 50 µg/mL Rituximab.

* Data shown here are from the Rituximab Innovator

Page 14: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab Nab 

2 Assays (Innovator and Biosimilar)

Assay format: Cell based

Binding?

Functional -- Go

Positive controls

Anti-Rituximab Id

Rabbit anti-Rituximab Polyclonal

Anti-Biosimilar Id

Nab Assay Development

Page 15: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab Nab 

Rituximab MOA:

Complement-dependent cytotoxicity (CDC)

Antibody-dependent cell-mediated cytotoxicity (ADCC)

ApoptosisA non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody (Gazzano-Santoro et. al., Journal

of Immunological Methods, 1997)

Nab Assay Development

http://minimednews.wordpress.com/

Page 16: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab Nab 

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

Nab Assay Development

Page 17: QPS Biosimilar Bioanalytical Approaches

Case Study: Rituximab Nab 

Nab Assay Development (Innovator)*

Validation Items

Approaches Results

Screening cut point(SCC)

At least 50 individual ST or RA lots

1. Floating cut point2. Significantly different SCPs

between matrixes3. Similar between Innovator and BSI

Immunodepletion At least 28 individual serum lots treated with Protein A/G

Similar between innovator and BSI

PC Crossing Check Anti-Biosimilar/innovator Abs in Innovator/BSI assay

PC showed similar activity in both innovator and BSI assays

Assay sensitivity Determined for all three positive control antibodies

Currently ongoing (~ 50 µg/mL)

Drug Tolerance Determined by High PC titering in the presence of various drug levels.

Can tolerate 10 µg/mL Rituximab at the estimated assay sensitivity

* Data shown here are from the Rituximab Innovator

Page 18: QPS Biosimilar Bioanalytical Approaches

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

Page 19: QPS Biosimilar Bioanalytical Approaches

Acknowledgement 

Joy HeFrank LiuLaurelle CallisteChristina XiaYun Shen

Avery TolosaYinLing LiBreann BarkerRoni Weaver

Jiannian ZhouMargaret Ma

LingSing ChenHui ZhangHolly ShenChad Asher