us pharma presentation on clone screen strategy for monoclonality using solentim cell metric cld

Efficient Screening Strategies to Address Monoclonality & Reduce TimelinesChaomei He, Associate Director , Momenta Pharmaceuticals, Inc. USAIBC 4th Annual Cell Line Development & Engineering Asia

Outline

Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved

Introducing Momenta Pharmaceuticals

Challenges in selection and clonal isolation: addressing

monoclonality

Review of latest technologies

Clonal cell lines selection using Cell Image System

Combining image processing and document tracking system for

effective clone-picking

Summary

Overview of Momenta Pharmaceuticals

Founded in 2001; IPO 2004

Located in Cambridge, Massachusetts, USA

~260 employees; >75% in scientific development

Expertise in high-resolution analytics, biological

characterization, and process engineering

Current business areas:

Complex drug generics

Biosimilars and potentially interchangeable biologics

Novel medicines

675 West Kendall StCambridge, MA

320 Bent StCambridge, MA


PROGRAMSPRECLINICAL/PROCESS

DEVELOPMENTCLINICAL1

ANDA/BLA/NDAFILED

MARKETED

ComplexGenerics1

Enoxaparin SodiumInjection (Generic Lovenox®)*

GlatopaTM (Generic 20 mg/mL Copaxone®)*

M356 (Generic 40 mg/mL Copaxone®)*

Biosimilars

M923 Adalimumab(HUMIRA®)**

M834 Abatacept (ORENCIA®)

M511 Bevacizumab (AVASTIN®)

6 Early Stage BiosimilarPrograms

NovelDrugs

Necuparanib - pancreaticcancer

hsIVIg (Hyper-sialylated IVIg)

SIF3 (Selective Immuno-modulator of Fc Receptors)

Anti-FcRn Antibody

Approved

ANDA Accepted

CTA Accepted/PK Study

Phase 2

1Clinical safety/efficacy trials have not been required for these complex generic drug applications

Momenta Product Portfolio: Broad Application of Thorough Characterization Across Complex Generics, Biosimilars, and Novel Drugs

*In collaboration with Sandoz**In collaboration with Baxter


- Vector Design

- Transfection

- Stable Pools

- 2-6 weeks

- Clones selection & screening- Scale up- Batch / Fed-Batch screening - 8-12 weeks

- Evaluation in bioreactors

- 4-6 weeks

- Cell Line Stability Study (Preliminary)

- 10-16 weeks

-Lead clone selection

-Generation of Research Cell Bank

A Typical Cell Line Development Flow Diagram for a Protein


Cell line development is a complex multistep process leading to the generation of a Research Cell Bank which is the precursor of Master Cell Bank: The properties of the selected clone will significantly impact the

process and the product itself Changing the cell line in the midst of a development program is

expensive and time consuming

Selection and Clonal Isolation


Proteins based therapeutic drugs have achieved great success in clinic, sales grew exponentially in the last decade, and this trend is expected to continue

Generation of clonal cell lines leading up to Research Cell Bank is typically the rate limiting step towards an IND

“The cell substrate is the transfected cells containing the desired sequences, which has been cloned from a single cell progenitor”ICH Guideline Q5D

Challenges in Streamlining Selection and Clonal Isolation


Efficient processes are essential to reduce timelines and costs Optimize each step from transfection to final clone by

defining criteria of success, implementing high throughput technologies and avoiding repeats. Build Quality in each step.

Documentation to support regulatory filings required from Day 1 From detailed information on the host cell line to

demonstration of monoclonality

Demonstration of monoclonality requires proper design, execution and documentation in the generation of the Research Cell Bank

Regulatory Requirements For Demonstration of Clonality

Anne B. Tolstrup – Boehringer Ingelheim Cell Line Development and Engineering, Berkeley 2014

How to Achieve Monoclonality ?

Two rounds of Limited Dilution Cloning (LDC) add significant time and expenses to the cell line development efforts

WCBP 2014 Sarah Kennett, Ph.D., FDA


How to Achieve Monoclonality ?

Use of a cell imaging system can reduce timelines and costs:

System needs to provide appropriate resolution and deliver documentation of the images

Operator needs to review images following wells from day zero Human intervention is required to ensure monoclonality

Significant IT requirements (huge files, need for high speed analysis, etc.)

WCBP 2014 Sarah Kennett, Ph.D., FDA


Latest Technologies For Single Cell Cloning

FACS AriaTM (BD Biosciences) Cloning by FACS at 1 cell/well

• Not all well has single cell, some well contain more than one cell

• Additional Cell image documentation is required to prove clonality

ClonePix FL System (Molecular devices), picking colonies in semi-solid medium

• Additional round of ClonePix is required to prove clonality

CloneSelectTM Imager (Molecular devices)

Cell MetricsTMCLD System (Solentim)

Octet High Throughput titer measurement (96, 384-well)


The Power of Imaging : Example of Single Colony Generated From a Single Cell Progenitor

Day 7

Day 3

Day 2

Day 1

Day 0


The Power of Imaging: Example of Single Colony Starting From Two Cells

Day 0 Day 1 Day 7


The Power of Imaging: Example of a False Negative

No cell was detected on day zero, but cells appeared in later days

No evidence that cells were derived from a single cell Resolution is critical

Day 0 Day 1

No cell


Percentage of Wells Contain Single Cell

Program Wells with Single Cell

Total # of Colonies

% Single Cell/ Total # of Colonies

Protein - A 783 1693 46%

Protein - B 721 2066 35%

Protein - C 436 1108 40%

Based on Poisson distribution, the probability of a single cell/well at seeding density of 0.5 cell/well dilution is 77%• In reality, the actual percentage of single cell/well is much lower

With a cell imaging system, day zero image can be captured• Select cell lines that demonstrate derivation from a single cell

progenitor The use of a cell imaging system allows for one round of limiting

dilution cloning, when performed using an appropriate dilution and acquiring images from day zero


Documentation of Monoclonality

Cell imaging system records original well ID of the single cell progenitor, and images associated with this well from day zero

An electronic automation tracking system can increase throughput and improve traceability• Well ID of the single cell progenitor - scale up - bioreactor – stability study

– lead clone selection – RCB – MCB/WCB

16 Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved

Example of Electronic Tracking

Record


Summary

Generation of a manufacturing cell line is typically the rate limiting step for filing an IND

The requirements for generating and testing manufacturing cell lines are well described in the literature

Demonstration of monoclonality is an important part of the overall Regulatory package

Monoclonality can be demonstrated by: 2 rounds of Limited Dilution Cloning

1 round of Limited Dilution Cloning with a cell imaging system

Cell Imaging system must have the appropriate resolution to allow for documentation of the initial doubling steps: from 1 cell to 2 cells to 4 cells.. Images need to be reviewed by operators

High demand on IT systems

Automation of well tracking increases throughput and improves traceability

Elimination of a Limited Dilution Cloning can reduce timelines significantly while meeting Regulatory requirements regarding monoclonality


Acknowledgments

LDC and Documentation

Billy Stuart Marcio Voloch

Megha Kapur


us pharma presentation on clone screen strategy for monoclonality using solentim cell metric cld

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