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Pu�ng Science to Work
Cell based ELISA approach for the es�ma�on of rela�ve potency of Kadcyla® (ado-trastuzumab emtansine)
M Y G A VM Y E A V
Pep�deMappingIntact
Mass Analysis
Glycosyla�on
AnitbodyDrug Conjugates
Stability
Host CellProteins
Iden�tyPurity & ChargeHeterogeneity
Centre for AdvancedProtein Studies
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HIGHLIGHTS
Pu�ng Science to Work
Syngene Interna�onal Ltd,Biocon Park, Plot 2&3, Bommasandra-Jigani Link Road, Bangalore 560 099, India.
Website: h�p://www.syngeneintl.com/services/biologics E-mail: [email protected]
@ Syngene Interna�onal Limited Syngene Interna�onal Limited@Syngeneintl
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An�body-drug conjugates (ADCs) are an emerging sub-class of biotherapeu�cs (magic bullet) offering dual advantage of having the specificity of mAbs and the cytotoxic payloads for targeted delivery of potent cytotoxic drugs to kill cancer cells. ADC’s are an inherently complex mixture of conjugated species, which differ in the number of payloads a�ached, as well as the a�achment sites of the payload on the mAb.
Kadcyla -Trastuzumab emtansine (also known as T-DM1) is a novel an�body–drug conjugate (ADC) that contains the humanized an�-HER2 IgG1 an�body trastuzumab and DM1, a microtubule inhibitory maytansinoid, linked through a thioether bond. Trastuzumab emtansine retains the mechanisms of ac�on of both, trastuzumab and DM1. Binding of T-DM1 to HER2 triggers the targeted entry of the HER2-T-DM1 complex into the cell via receptor-mediated endocytosis. The proteoly�c degrada�on of the an�body part of T-DM1 within the lysosome, results in the release of ac�ve DM1 which inhibits microtubule assembly, causing the arrest of the cells in the G2-M phase of cell cycle, resul�ng in apopto�c cell death.
In 2013, FDA approved ado-trastuzumab emtansine (Kadcyla) for the treatment of human epidermal growth factor receptor (HER)2- posi�ve breast cancer. Kadcyla consists of the therapeu�c an�-HER2 an�body trastuzumab, which is marketed under the brand name Hercep�n and which is covalently linked to a maytansine deriva�ve (DM1) via a linker (Fig. 1). Kadcyla is a lysine-conjugated ADC, it u�lizes the solvent-exposed e-amino groups of lysine residues to a�ach drugs.
Figure 1. Schematic representation of Kadcyla and Herceptin.
We have used a Cell based ELISA (Enzyme Linked Immunosorbent Assay) approach for the es�ma�on of the rela�ve potency of Kadcyla.
INTRODUCTION
SPECIFICITY
LINEARITY
PRECISIONKadcyla binds to the HER2 receptor, which is expressed onto the BT474 cells. The bound drug is detected using a peroxidase conjugated an� human IgG an�body and quan�fied using TMB substrate. The intensity of colour is directly propor�onal to the concentra�on of bound drug.
BT-474 cells expressing HER-2 receptor were seeded on a 96 well plate. The cells were fixed on the plate using a fixa�ve. The binding capacity of Kadcyla was measured over a 0.05 µg/mL to 0.0006 µg/mL concentra�on range. Es�ma�on of rela�ve potency was done using 4PL analysis in So�Max Pro so�ware.
Intra assay precision of the method was calculated as the % RSD for potency values of 100 % Kadcyla (n=4). Inter assay precision was calculated as the % RSD for potency values at each of the concentra�on levels; 64%, 80%, 120% and 156% (n=6) and for 100% (n=8).
The Regression (R2) value for the linear plot of observed potency of individual determinants with theore�cal potency was found to be 0.999 and the method is linear over the range of 64% – 156 % of 100 % Kadcyla.
Intra and inter assay precision was found to be NMT 4.6% for different concentra�ons of Kadcyla and the method was precise for es�ma�ng the rela�ve potency (in %) of Kadcyla by Cell based ELISA.
The specificity of the assay was checked using a formula�on buffer and a non-specific mAb, like Ipilimumab. The formula�on buffer and Ipilimumab were tested at the same dose range as that of Kadcyla.
Linearity and range of the method was evaluated by comparing the observed potency with the theore�cal potency at each of the concentra�on levels (64, 80, 100, 120 and 156%) for Kadcyla. A linear response was generated by using the linear regression model, y = mx + c.
CONCLUSIONThe bioassay for the determina�on of rela�ve
potency of ado-trastuzumab emtansine was developed and qualified. The assay is suitable to use for its intended purpose of batch release and stability analysis.
No significant response was observed with either formula�on buffer or Ipilimumab, demonstra�ng the specificity of the assay for Kadcyla.
Figure 2: Representative 4PL sigmoidal curve of Kadcyla sample analysis using global fit for potency estimation
Table 1: Intra and Inter Assay Precision of assay
Figure 3: Specificity analysis of assay for Kadcyla using normal saline and Ipilimumab
Figure 4: Linear curve plotted between theoretical potency Vs. observed potency of Kadcyla.
Table 2: Statistical Parameters for linearity data
INTER ASSAY PRECISION
INTRA ASSAY PRECISION
Theore�cal Potency (%) Mean RP (%) SD %CV
Theore�cal Potency (%) Mean RP (%) SD %CV
64% (n=6)
80% (n=6)
100% (n=8)
120% (n=6)
156%(n=6)
66%
79%
101%
121%
155%
2.6
1.8
4.6
4.1
3.1
3.9
2.3
4.6
3.4
2.0
100% (n=8) 102% 3.7 3.6
Y intercept 2.327
Slope 0.981
R2 0.999
STATISTICAL PARAMETERS
REFERENCES• Trastuzumab emtansine: mechanism of ac�on and drug resistance, Breast Cancer Res. 2014; 16(2): 209.
• T-DM1, a novel an�body–drug conjugate, is highly effec�ve against primary HER2 over expressing uterine serous carcinoma in vitro and in vivo, Cancer Med. 2014; 3(5): 1256-65.
h�ps://www.accessdata.fda.gov/drugsa�da_docs/label/2013/125427lbl.pdf•
