Thesis Final

1 | P a g e

A project report on

ANALYSIS OF BIOLOGICAL ACTIVITY OF RECOMBINANT

THERAPUTIC BIOLOGICS

Submitted to

Biotechnology Consortium India Limited (BCIL)

Department of Biotechnology,

Government of India, New Delhi-110002

Submitted by

RAJANA VINOD KUMAR

For fulfilment of Biotech Industrial Training Programme (2014-15)

Under the supervision of

Mrs. SHUBHANGI ARGADE

Sr. Research Scientist, R&D

Preformed At

INTAS Pharmaceuticals Ltd. – Biopharma Division

Plot Number 423/P/A-GIDC, Sarkhej- Bavla Highway, Moraiya,

Ahmedabad, Gujarat

2 | P a g e

DECLARATION

I hereby state that, to the best of my knowledge and belief, the project report entitled “Analysis

of biological activity of recombinant therapeutics biologics” being submitted carried out under

the supervision of Mrs. Shubhangi Argade, Sr. Research Scientist, R&D, Pharmaceuticals Ltd. –

Biopharma Division, Ahmedabad. I assure that this data is from my practical experience and skill

development, will not be published, represented or used for any other purpose in future. I abide

that all the data presented in this report will be treated with utmost confidentiality.

Date: 08/05/15 Rajana Vinod Kumar

3 | P a g e

ACKNOWLEDGEMENTOn the verge of project work, I take the opportunity to express my deep sense of gratitude,

heartfelt thanks and indebtedness to all those who have directly or in directly helped me in my

work.

My first and foremost thanks to Biotech Consortium India Ltd, Department of Biotechnology,

New Delhi, for providing me an opportunity of a lifetime to work at Intas Biopharmaceuticals

limited, Ahmedabad.

I would like to thank Mr. Anuj Shah (Deputy General Manger, HR) and Mr. Ajit More

(Executive, HR) for giving me a chance to do this training.

I would like to acknowledge Dr. Gargi Sheth for granting me an opportunity to work in BDL at

Intas Pharmaceuticals Ltd. – Biopharma Division and for giving such a great start to my career.

I am out of words to thank my guide, Mrs. Shubhangi Argade, BDL for training and mentoring

me. She solved every bit of my query very patiently. She has been a constant guiding light

during my project.

I would like to say my special thanks to Mr. Suvas kumar Patel, Mrs Nikita Patel for their

utmost care and patience to teach me each and every aspect related to my quarries’ during

training

I would also like to acknowledge Dr. Jayesh Maradiya, Mrs. Poonam Kapoor, Mr. Saurabh

Pathak, Mr. Milan Vachhani, Ms Hemangini Chowdhary, Mrs. Prutha Patel, Ms Chinar Shah,

Ms Ruchika Sharma, Ms. Ritika Sutariya, Ms. Tanha Rana, Mrs. Shweta Bheda, Mr. Somnath

Gupta, Mr. Harshil Sanghani, Mr. Sunny Trivedi, BDL staff and INTAS staff for imparting

knowledge about something which was very new to me and supporting and encouraging

throughout the coursework.

Last but not least, I would like to thank my parents for their endless support, patience and care.

4 | P a g e

CONTENT

Introduction………………………………………………………………….05

Aim & Objective…………………………………….………………………08

Materials and method………………………………………………………..09

Techniques/databases used …………………………………………….……20

Equipments handled……………………………...………………………….24

Results……………………………………………………………………….26

Conclusion ………………………………………………...………………..39

5 | P a g e

INTRODUCTION

6 | P a g e

INTRODUCTION

RECOMBINANT BIOTHERAPEUTIC PROTEINS are proteins that are engineered in the

laboratory for pharmaceutical use; in specific host cell (bacteria, yeast, mammalian) under

defined conditions by Recombinant DNA technology.

Therapeutic proteins include; Interferon’s, Cytokines (rGCSF), Enzymes (rtpA) Clotting factors

(rFactorIX,), Monoclonal antibodies (Mabs), Anticoagulant (rHirudin) Hormones (rFSH,) etc.

Out of which monoclonal antibodies are fastest growing class of therapeutic proteins.

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are made by identical

immune cells. There are three types of monoclonal antibodies

Chimeric antibodies have murine variable region and constant region of human antibody

Humanized antibodies have only CDR is mouse origin

Human monoclonal antibodies are fully human

Monoclonal antibodies are engineered based on primary classes of immunoglobulins are IgG,

IgM, IgA, IgD and IgE. Among these IgG is widely distributed in the blood and tissue. In

humans there are four subclasses of IgG viz. IgG1, IgG2, IgG3 and IgG4 (numbered in order of

decreasing concentration in serum).

The monoclonal antibody in my present study is a IgG2 type monoclonal antibody

A full human anti-RANKL IgG2 monoclonal antibody, that binds to human RANKL with high

affinity and mimics endogenous osteoprotegerin (OPG). It is produced in chinese hamster ovary

(CHO) cells. RANKL bind to RANK receptor (receptor activator NF-kB) present on

preosteoclast and induced osteoclastogenesis and bone resorption. Anti-RANKL IgG2

monoclonal antibody inhibits both membrane bound and soluble primate RANKL lead to

reduction in resorption and increases bone mineral density (BMD) and strength.

Biotherapeutics need characterization before marketing. Protein for therapeutic application

demands highly sensitive and specific analytical methods. It should be identical to that of natural

protein in its biochemical and functional properties and so it should be well characterized where

its identity, purity, impurities, quantity, stability, potency and quality can be determined and

7 | P a g e

controlled. As these are large and complex molecules their characterization demands an array of

analytical techniques. Characterization also set limits of assays for the assurance of these limits.

Analytical techniques used for characterization like SDS-PAGE, chromatography, peptide

mapping, western blot, iso electric focusing, and bioassays etc.

Bioassay is an assay used to determine the potency or biological activity of a substance, In-vivo

(living organisms) and in- vitro assays cell based & ELISA (enzyme linked immunosorbent

assay).

This study deals with Anti-RANKL IgG2 monoclonal antibody binding efficacy to the RANKL

by solid phase binding assay. And its RANKL neutralizing capacity by cell based assay using

RAW264.7 cell lines. Since the bioassay involves many dilution steps and are key for the final

results; various experiments were carried out to evaluate the pipetting technique skills.

8 | P a g e

AIM AND OBJECTIVE

9 | P a g e

AIM and OBJECTIVE:-

AIM: -

Analysis of biological activity of recombinant therapeutic biologic- Anti-RANKL IgG2

monoclonal antibody.

OBJECTIVE:-

Analysis of binding efficiency and RANKL neutralizing activity of Anti-RANKL IgG2

monoclonal antibody by binding assay and cell based assay.

10 | P a g e

MATERIALS AND METHODS

11 | P a g e

MATERIALS:-

sRANKL

Sodium carbonate anhydrous (Na2CO3)

Sodium bicarbonate (NaHCO3)

Sodium chloride (NaCl)

Sodium phosphate dibasic (Na2HPO4)

Potassium phosphate Monobasic (KH2PO4)

Potassium chloride (KCl)

Bovine Serum Albumin (BSA)

Polysorbate-20 (Tween-20)

Concentrated H2SO4

Peroxidase Conjugated protein-A

TMB/H2O2

Maxisorp Strips

Water for injection (WFI)

Micro centrifuge tubes

Reagent Reservoir

96 well plates

Tissue culture flasks (TCF)

Serological pipettes

Micro pipettes – single channel and multichannel ( manual and electronic)

Preparation of 0.05% amido black solution:-

Dissolve 4.1 g sodium acetate in 500 mL water

Take 455 mL of it & add 45 mL of the glacial acetic acid.

Dissolve 0.250 g of the amido black powder into 500mL solution

Growth media:-

DMEM high glucose

FBS

12 | P a g e

Sodium pyruvate

Antibiotic – antimycotic solution

Mix all components aseptically and vacuum filter it through 0.22µm sterile filter. Store at 2-8 ˚C

for cell maintenance

Assay media:-

MEM alpha

FBS

Antibiotic-antimycotic solution

Mix all components aseptically and vacuum filter it through 0.22µm sterile filter. Store at 2-8 ˚C

for assay

2M citric acid

Citric acid anhydrate…………………. 19.21g

Water for injection……………………..50mL

Mix and store at RT

20mM citrate buffer

Trisodium citrate dehydrate…………….1.47 g

Water For Injection (Quantity sufficient).100mL

Dissolve in 80mL of WFI adjust pH to 4.8±0.2 with 2M citric acid and then makeup to 100 mL

0.1N NAOH

NaOH (sodium hydroxide)……………….400g

Water For Injection (Quantity sufficient)...100mL

Mix and store at RT

10%v/v Fixing solution (freshly prepared)

Substrate solution

13 | P a g e

di- sodium tartrate dihydrate…………..….23 mg

(pNPP) para-Nitro phenyl phosphate……..18.6 mg

20mM citrate buffer……………………….10 mL

Mix in light protected container

14 | P a g e

METHODS

Experiment:-1

Objective: - Dispensing of 50 µl & 100 µl amido black solution into 96-well plate using

multichannel pipette

Procedure of dispensing 50µl amido black solution

Dilute 25 mL of amido black solution into 225 mL water for injection

Dispense horizontally 50 µl amido black using multichannel pipette in 96well plate

Gentle tap each plate & Read out at OD 630 nm

Procedure of Dispensing 100 µl amido black solution


Dispense horizontally 100 µl amido black using multichannel pipette in 96well plate


Experiment:-2Objective: - To Practice serial Dilution using amido black in 96-well plate: Set-1[100µl, 6+6

Channel], Set-2[100µl , 3+3+3+3 Channel ], Set-3[100µl , 8+4 Channel ],

Procedure for Serial Dilution

Take 96 well plate and label them

Add 100 µl of WFI in the entire plate except the first row using multichannel pipette in

set-1[6+6], set-2[3+3+3+3] and set-3[8+4] plate pattern.

Fill first row with 200µl of 0.05% amido black.

Then take 100 µl of amido black solution from the first row and add to the second row,

mix properly & again repeat steps up to last row.

From the last row after mixing properly take 100 µl of the solution & discard.

Read absorbance at OD 630nm.

15 | P a g e

Experiment:-3

Objective: - Dispensing of 100 µl amido black solution into 96-well plate using Electronic

Pipetting

Procedure of Dispensing 100 µl amido black solution


Dispense horizontally 100 µl amido black using electronic pipette in 96well plate


Experiment:-4

Objective: - To Practice serial Dilution of 100µl amido black in 96-well plate,[ 6+6 ],

[3+3+3+3],[8+4] electronic pipette

Procedure for Serial Dilution

Take 96 well plate and label them

Add 100 µl of WFI in the entire plate except the first row using electronic pipette in plate

A[6+6], plate B[3+3+3+3] and plate C[8+4]

Fill first row with 200µl of 0.05% amido black.

Then take 100 µl of amido black solution from the first row and add to the second row,

mix properly & again repeat steps up to last row.

From the last row after mixing properly take 100 µl of the solution & discard.

Read absorbance at OD 630nm.

Experiment:-5

Objective: - To analyze binding efficiency of anti-RANKL antibody by Solid Phase binding

assay and to check the assessment of accuracy and precision.

Principle:-

16 | P a g e

The binding activity of Anti-RANKL IgG2 monoclonal antibody is determined by the solid

phase binding assay by using soluble RANKL as antigen. 96well plate is coated with soluble

RANKL and incubated at different concentration of Anti-RANKL IgG2 monoclonal antibody

reference standard and test sample. Protein-A labeled with horse radish peroxidase (HRP)

enzyme is used as secondary antibody to determine binding of Anti-RANKL IgG2 monoclonal

antibody to sRANKL by colorimetric reaction on addition of TMB (3,3’,5,5’-

tetramethylbenzidine).

TMB is a chromogen yields blue color when oxidized with hydrogen peroxide, catalyzed by

HRP. By the addition of stop solution (0.1N H2SO4) blue color changes to yellow. Absorbance

read at 450 nm.

Binding of test sample is determined by comparing response with reference standard.

Procedure for binding assay:-

Coating of antigen: - 100 μl of 75 ng/mL of RANKL was coated on ELISA plate.

Incubation :- Incubation at 25 °C and 100 rpm for 2 hour

Washing-1 :- Wash the plate with 300 μl wash buffer (PBST)three times

Blocking :- 300 μl of blocking solution (BSA+PBST)

Washing-2:- Wash the plate with 300 μl wash buffer (PBS+TWEEN-20)

Addition of standard and sample

Dilution of standard solution and sample (1125ng/mL to 0.703 ng/mL)

Incubation:- Incubated for 2 hours

Washing-3:- Wash the plate with 300 μl wash buffer (PBS+TWEEN-20)

Detection antibody preparation: - 20,000 fold diluted Pro-A HRP 100µl of secondary

antibody is added.

Incubation:- Incubated at 25 °C and 100 rpm for 30 minutes

Washing-4 :- Wash the plate with 300 μl wash buffer (PBS+TWEEN-20)

Substrate preparation (TMB) and addition; 1:20 fold diluted TMB in WFI. 100 µl is

added.

Incubation:- Incubate at 25 °C and 100 rpm for 30 minutes

17 | P a g e

Add stop solution:- Immediately after 30 minutes of incubation, 100 μl of stop

solution( 0.1N H2SO4 ) is added

Absorbance read at 450 nm.

Potency Calculation

Feed the absorbance obtained from Standard and sample to PLA software

Use linear fit model and analyze potency in comparison to standard

Check for linearity, regression and parallelism and report the % potency

Experiment:-6

Objective: - To analyze neutralizing activity of anti-RANKL antibody by Cell based assay.

Sub Culture And Cell Counting Of Cells:-

1. Remove media from the flask

2. Add 5mL PBS without disturbing cell layer at bottom surface of flask.

3. Make it spread all over the cell growth surface

4. Then remove PBS and add 0.5 mL trypsin 0.5%

5. Incubate it for 2-3 min and tap it gently.

6. Then add 4.5mL DMEM growth medium

7. Total volume of T-25 flask is 5mL

8. Now subculture it in new flask and add volume of medium according to the cell count

and required density

Formula for cell volume calculation:-

Cell volume for desired density = required density × required volume ÷cell count

Cell counting

Principle:. The mechanism of trypan blue staining is based on it being negatively charged and

not interacting with cells unless the membrane is damaged.. Indeed, undamaged cells are very

selective concerning the compounds that pass through their membrane, and thus should not take

up trypan blue. Therefore, all the cells that exclude the dye are considered viable.

18 | P a g e

ProcedureSample dilution preparation

Cells sample was diluted with PBS in aliquots according to required ratios i.e. (1:2)

(1:5) (1:10) including trypan blue for viable cell count.

First slide was cleaned with WFI & dried with gauze piece

Sample were taken from different dilution sample tubes & loaded in hemocytometers

Arrange the hemocytometer into the inverted microscope and observe the cell into

10x magnifications of the inverted microscope and count was taken in 4 square

Calculate the total number of viable and dead cell in each chamber.

Calculate the average of the total number of cells.

Calculation

Cell count =Average number of cells *Dilution factor ×104 Cell/mL

Viable count = Average number of viable cells* dilution factor × 104 Cell/mL

Non-Viable count = Average number of non-viable cell *dilution factor × 104

Cell/mL

Total Count =Viable Cells + Non-Viable cells

% Viability = Viable cell count ×100/Total cell count

PRINCIPLE OF CELL BASED ASSAY:-

RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived

from BALB/c mice have RANK receptor.

The biological activity Anti-RANKL IgG2 monoclonal antibody is determined in a cell based

bioassay using RAW 264.7. Upon stimulation with RANKL, the cells undergo

osteoclastogenesis which is indicated by detection of TRAP enzyme. Anti-RANKL IgG2

monoclonal antibody specifically binds to RANKL and inhibits its stimulatory effect on cells in

a dose dependent manner. The cells are incubated with fixed amount of RANKL and varying

concentrations of anti-RANKL IgG2 monoclonal antibody reference solution and test solution.

After a timed incubation, cells are fixed and incubated with pNPP in presence of sodium tartrate.

The cellular TRAP enzymes will convert the substrate to p-Nitrophenol which is further

converted to yellow colored p-nitrophenolate in alkaline condition that is measured at 405 nm.

19 | P a g e

The absorbance value is directly proportional to the number of osteoclasts in culture and

inversely to the concentration of anti-RANKL IgG2 monoclonal antibody applied. Relative

potency of test sample is determined by comparing its dose response to that of reference

solution.

Procedure for cell based assay

Add 10000 cells/well to plate and keep at RT for1hr

Standard and test doses dilutions were prepared using dose range 400 ng/mL to 0.66

ng/mL, and add of 50 µl of standard and test doses to the plate and 50 µl of RANKL in

each well.

Incubate for 72 hr in CO2 incubator

TRAP assay

After 72 hr remove the media with PBS wash

Fix the cells by fixing solution (formaldehyde solution) and incubate for 10 min

Remove fixing solution by PBS wash

Add pNPP and tartarate solution 100 µl in each well under dark condition and incubate

for 1 hr

Add 50 µl of 0.1N NaOH solution in new plate

Transfer supernatant from pNPP tartarate solution into NaOH new plate and read

absorbance at 405 nm

Potency Calculation

Feed the absorbance obtained from Standard and sample to PLA software

Use linear fit model and anlayse potency in comparison to standard

Check for linearity, regression and parallelism and report the % potency

20 | P a g e

TECHNIQUES AND DATABASE

EnSpire Multimode Plate Reader:-

21 | P a g e

EnSpire® Multimode Plate Reader is designed to meet the demands of multi-user research

environments, providing high performance technology and flexibility for a range of applications

without compromising sensitivity, PerkinElmer offers an unrivaled solution to application needs.

a flexible and upgradeable multimode plate reader which includes the Absorbance mode as standard.

Integrated data analysis software with data export (Excel® or text files) features

Ultra-sensitive luminescence option for detecting fewer cells than standard luminescence,

ideal for primary, stem or difficult-to-transfect cells

Quad-monochromator for fluorescence and absorbance – select any wavelength for the

best possible signal to background for fluorescence applications

Time-resolved fluorescence provides high sensitivity for biological and cellular assays.

Make the most of your high-quality data generated using the EnSpire multimode plate reader by

harnessing the advanced data visualization and analysis capabilities the analysis of plate reader

data and enables you to visualize, correlate, compare and analyze your data.

SOFTMAX PRO V5.3

22 | P a g e

SoftMax pro V5.3 display

SoftMax pro 5.3 from molecular devices offers extensive data capture, analysis, &

reduction capabilities for its absorbance, luminescence, fluorescence & multi-detection

micro plate readers for customers working in pharmaceuticals, biotechnology, academic,

and hospital or government laboratories. It possesses the property of customizable read

through robotic integrations. It is assigned with enhanced curve fitting for complex data

sets comprising 4 and 5 parameter logistic curve fits. It can also perform analysis of EC50

calculations and tests for parallelism, relative potency, non linear confidence intervals and

statistics. The reader supports automatic flagging of results through conditional formulas.

PLA software:-

23 | P a g e

PLA is the commercial solution for assay analysis with the parallel-line and parallel-logistics

methods. Proven statistics for the easy execution of parallel-line and parallel logistics analytics

PLA 2.0 is easy to learn, easy to use and to deploy

Extended analysis functionality. The program finds linear and parallel regions in assays

automatically. You can fine-tune this feature with many available program options.

Highlights:-

Difference tests (F-Tests) and similarity testing (equivalence tests)

Mix difference testing and similarity testing in the same assay (optional)

32 different tests available - better control over the tests in your assay systems

Develop equivalence margins for similarity testing with PLA 2.1

Speed: The statistics core of PLA is highly optimized. Depending on the number of

measurements, the analysis normally takes only a few seconds to complete

PLA2.0 software display:-

24 | P a g e

EQUIPMENTS HANDLED

25 | P a g e

EQUIPMENTS/INSTRUMENTS HANDLED:-

Single Channel Pipette Thermo scientific

Multi Channel Pipette Thermo scientific

Electronic multi channel pipette Brand, Thermo Scientific

Weighing balance Sartorius

pH meter LABINDIA

Vortexer Gilson

Microplate shaker Shell Lab

Microscope Columbus

Biosafety hood ESCO

CO2 incubator Shell Biolabs

Waterbath Scescy

Automated cell counter Countless

Centirfuge Ependroff

Multi Mode plate reader PerkinElmer

SoftMax Pro software Molecular Devices

PLA 2.0 Software Stegmann Systems Germany

26 | P a g e

RESULTS

27 | P a g e

Experiment: - 1

Dispensing of 50 µl amido black solution into 96-well plate

(Multi Channel)

Plate A:-

Plate A Plate B Plate C

Overall

Avg0.38 0.24 0.32

Overall

Stdev0.02 0.02 0.02

Over all

%CV5.64 9.20 7.48

Dispensing of 100 µl amido black solution into 96-well plate

(Multi Channel)

Plate A:-


Overall

Avg0.67 0.68 0.68

Overall

Stdev0.02 0.03 0.02

Overall

%CV3.29 3.85 3.38

28 | P a g e

Experiment:-2 To Practice serial Dilution of amido black in 96-well plate set-1 [100µl, 6+6 Channel]

Plate A:-

Set A1:- Set A2:-

Avg Stdev %Cv

2.38 0.06 2.58

1.70 0.01 0.57

0.93 0.03 3.27

0.54 0.02 3.60

0.28 0.01 5.08

0.17 0.00 2.68

0.10 0.00 2.60

0.06 0.00 3.83

Plate B:-

Set B1:- Set B2:-

Avg Stdev %CV2.23 0.02 0.69

1.70 0.04 2.44

0.94 0.06 6.80

0.56 0.03 5.33

0.27 0.02 6.68

0.18 0.01 4.56

0.10 0.01 7.82

0.07 0.00 1.88

Avg Stdev %CV2.40 0.06 2.60

1.64 0.03 2.09

0.92 0.03 3.63

0.47 0.02 4.09

0.26 0.01 2.46

0.15 0.00 1.19

0.09 0.00 3.71

0.06 0.00 2.28

Avg Stdev %CV2.23 0.03 1.39

1.69 0.06 3.26

0.97 0.04 3.93

0.52 0.03 5.75

0.29 0.01 2.58

0.17 0.01 4.60

0.10 0.00 1.69

0.07 0.00 5.56

29 | P a g e

Plate C:-

Set C1:- Set C2:-

Avg Stdev %CV

2.75 0.17 6.03

1.92 0.04 2.02

0.99 0.03 2.79

0.57 0.02 3.22

0.29 0.01 3.56

0.17 0.02 13.33

0.10 0.00 2.91

0.08 0.00 4.63

To Practice serial Dilution of amido black in 96-well plate set -2 [100µl, 3+3+3+3 Channel]

Plate A:-

Set A1 Set A2 Set A3 Set A4 Avg Stdev %CV

2.05 1.97 2.07 2.03 2.03 0.04 2.03

1.13 1.11 1.10 1.12 1.12 0.01 1.10

0.57 0.59 0.58 0.58 0.58 0.01 1.17

0.34 0.33 0.32 0.33 0.33 0.01 2.12

0.18 0.18 0.18 0.18 0.18 0.00 0.83

0.13 0.12 0.11 0.12 0.12 0.01 6.20

0.08 0.07 0.07 0.07 0.07 0.00 3.66

0.06 0.06 0.06 0.06 0.06 0.00 1.09

Avg Stdev %CV

2.73 0.13 4.63

1.77 0.06 3.44

0.96 0.02 2.02

0.51 0.02 4.45

0.29 0.01 3.37

0.16 0.01 4.87

0.10 0.00 1.73

0.07 0.00 6.53

30 | P a g e

Plate B:-

Set B1 Set B2 Set B3 Set B4 Avg Stdev %CV

2.06 2.10 2.09 2.12 2.09 0.02 1.17

1.12 1.11 1.10 1.08 1.10 0.02 1.44

0.57 0.57 0.60 0.58 0.58 0.01 2.43

0.34 0.32 0.32 0.31 0.32 0.01 3.99

0.18 0.17 0.17 0.18 0.17 0.00 2.26

0.11 0.11 0.10 0.11 0.11 0.00 3.02

0.07 0.07 0.07 0.07 0.07 0.00 1.32

Plate C:-

Set C1 Set C2 Set C3 Set C4 Avg Stdev %CV2.12 2.20 2.22 2.19 2.18 0.05 2.08

1.13 1.15 1.11 1.09 1.12 0.03 2.31

0.60 0.60 0.61 0.61 0.60 0.00 0.77

0.33 0.33 0.33 0.32 0.33 0.01 2.48

0.18 0.18 0.19 0.18 0.18 0.01 2.82

0.12 0.11 0.11 0.11 0.11 0.00 3.24

0.07 0.07 0.07 0.07 0.07 0.00 0.59

0.05 0.05 0.05 0.06 0.05 0.00 1.04

31 | P a g e

To Practice serial Dilution of amido black in 96-well plate set-3 [100µl, 8+4 Channel]

Plate A Set A1 Set A2 Avg Stdev %CV

2.03 2.07 2.05 0.03 1.44

1.12 1.08 1.10 0.03 2.68

0.57 0.60 0.58 0.02 3.21

0.34 0.31 0.32 0.02 5.52

0.18 0.18 0.18 0.01 2.90

0.11 0.11 0.11 0.00 0.97

0.07 0.07 0.07 0.00 1.60

0.05 0.06 0.06 0.00 2.54

Plate B Set B1 Set B2 Avg Stdev %CV

2.14 2.13 2.14 0.01 0.43

1.11 1.09 1.10 0.01 1.06

0.56 0.59 0.58 0.02 2.72

0.32 0.31 0.32 0.01 2.10

0.17 0.19 0.18 0.01 7.41

0.11 0.11 0.11 0.00 0.49

0.07 0.07 0.07 0.00 0.90

0.05 0.05 0.05 0.00 0.82

Plate C Set C1 Set C2 Avg Stdev %CV

2.07 2.10 2.08 0.02 1.04

1.13 1.08 1.11 0.04 3.37

0.57 0.59 0.58 0.01 2.32

0.32 0.31 0.31 0.00 1.46

0.17 0.18 0.17 0.01 3.34

0.11 0.10 0.10 0.01 7.08

0.07 0.07 0.07 0.00 4.30

0.06 0.05 0.05 0.00 0.97

32 | P a g e

Experiment:-3Dispensing of 100 µl amido black solution into 96-well plate

(ELECTRONIC PIPETTING)

Plate A:-


Overall

Avg0.75 0.75 0.75

Overall

Stdev0.03 0.03 0.02

Overall

%CV3.55 3.60 3.10

Experiment:-4RESULT: - To Practice serial Dilution of 100µl amido black in 96-well plate,[ 6+6 ],

[3+3+3+3],[8+4] electronic pipette

Plate

6+6 Avg Stdev %CV Avg Stdev %CV

3.36 0.30 9.01 3.38 0.32 9.46

1.39 0.02 1.11 1.49 0.03 2.32

0.80 0.03 3.71 0.22 0.08 35.31

0.47 0.01 2.92 0.52 0.03 6.67

0.23 0.01 2.24 0.36 0.02 5.09

0.15 0.00 2.43 0.24 0.01 5.67

0.09 0.00 4.82 0.16 0.01 7.93

0.06 0.00 2.99 0.17 0.02 10.79

33 | P a g e

Plate

3+3 Avg Stdev %CV Avg Stdev %CV0.31 0.20 64.77 2.86 0.09 3.17

1.84 0.28 15.41 1.87 0.10 5.37

1.24 0.02 1.49 0.97 0.03 2.64

0.86 0.05 5.34 0.59 0.02 2.77

0.51 0.04 7.47 0.30 0.01 3.57

0.56 0.02 4.00 0.18 0.00 2.20

0.18 0.01 4.25 0.10 0.00 1.02

0.11 0.01 4.93 0.07 0.00 0.80

Avg Stdev %CV Avg Stdev %CV

2.74 0.11 4.01 2.86 0.09 3.17

1.77 0.07 3.95 1.83 0.07 3.91

1.02 0.04 3.78 1.01 0.04 4.05

0.55 0.02 3.83 0.57 0.03 4.74

0.33 0.01 3.14 0.32 0.02 5.18

0.17 0.01 3.26 0.18 0.01 2.97

0.10 0.00 3.86 0.11 0.01 6.58

0.07 0.00 1.37 0.07 0.00 4.75

34 | P a g e

Plate

8+4 Avg Stdev %CV Avg Stdev %CV

2.75 0.12 4.35 2.73 0.10 3.81

1.89 0.04 2.08 1.71 0.06 3.37

1.02 0.02 2.24 0.97 0.02 2.43

0.57 0.01 1.67 0.52 0.01 1.27

0.30 0.01 2.42 0.30 0.01 4.90

0.17 0.01 3.17 0.17 0.00 1.93

0.10 0.00 2.26 0.10 0.00 3.36

0.07 0.00 1.23 0.06 0.00 3.51

35 | P a g e

Experiment 5:-

RESULT: - SOLID PHASE BINDING ASSAY

x axis

0.1 1 10 100 1000 100000

0.5

1

1.5

2

Experiment#05

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Standard (Standard: Concentration vs MeanValue) 0.0851 1.08 18.2 2.04 0.999

__________Curve Fit Option - Fixed Weight Value

36 | P a g e

ACCURACY ASSAY:-

sample % relative potency % Recovery

Sample 1 (60%) 63 105

Sample 2 (80%) 79 99

Sample 3 (100%) 100 100

Sample 4 (130%) 138 106

Sample 5 (160%) 175 109

log concentration of Denosumab

0.1 1 10 100 1000 100000

0.5

1

1.5

2

2.5P23 Binding Assay-A

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Standard (Standard: Concentration vs MeanVal... 0.113 1.29 16.8 2.11 0.997100% (Test sample 3: Concentration vs MeanV... 0.0756 1.07 20.3 2.35 0.99960% (Test sample 1: Concentration vs MeanVal... 0.09 1.18 27.1 2.12 0.99980% (Test sample 2: Concentration vs MeanVal... 0.0735 1.08 24.8 2.29 1


37 | P a g e

Experiment 6:-

RESULT OF CELL COUNTING

log concentration of Denosumab

0.1 1 10 100 1000 100000

0.5

1

1.5

2

2.5P23 Binding Assay-B

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Standard (Standard: Concentration vs MeanVal... 0.142 1.25 14.8 1.86 0.997130% (Test sample 1: Concentration vs MeanV... 0.106 1.09 10.8 1.89 0.999160% (Test sample 2: Concentration vs MeanV... 0.0705 0.962 9.62 2.03 1


total count

0.84x10^6

0.94x10^6

0.19x10^6

1.03x10^6

1.08x10^6

1.31 x 10^6

Averag

e

1.06x10^6

Std 0.16

%CV 15.89

total count

0.20x10^6

0.22x10^6

0.26x10^6

0.31x10^6

0.32x10^6

0.33x10^6

Averag

e

0.27x10^6

Stdev 0.05

%CV 20.12

38 | P a g e

RESULT: - METHOD FOR CELL BASED ASSAY

sample % relative potency

test solution 1 98

Test solution 2 102

Average Potency 100

% CV 3

Log Concentration (ng/mL)

0.1 1 10 100 1000 10000

0

0.1

0.2

0.3

0.4

0.5

0.6P23 4PL Curve Fit-A

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2P23U36D1 (Reference Solution: Concentration vs ... 0.548 1.9 5.62 -0.000374 0.997P23U36D1 (Reference Solution: Concentration vs ... 0.548 1.9 5.62 -0.000374 0.997P23U36D1 (Test Solution 2: Concentration vs Mean... 0.522 1.94 5.95 -0.000765 0.997


39 | P a g e

CONCLUSION

40 | P a g e

Conclusion:-The first and foremost requirement for the characterization of the bio therapeutics biologics

through bioassay requires efficiency in pipette handling. Hence; my primary experiments are

related to the handling efficiency of different volumes by manual and electronic pipette. The

efficiency was determined based upon the % coefficient of variance (%CV). As the % cv is

within the expected limits of < 10%; dilution experiments were concluded as pass.

The solid phase binding assay yielded dose dependant sigmoid dose response curve with clear

upper and lower asymptote. This proved the binding efficiency of anti-RANKL antibody to its

ligand RANKL.

The accuracy experiment results showed expected potency values compared to standard and

recovery was within desired range of ± 10%. The analysis is accurate and precise.

Ïn cell based assay test material was tested against standard in two independent analylsis. The

average potency obtained was 100 % with 3 % CV.

Biological activity of anti-RANKL antibody by two different bioassays was carried out and

material was found to be of same potency as that of standard.

Thesis Final

Documents