CHALLENGES IN PROCESS DEVELOPMENT, SCALE UP ... · PDF fileBiologics World Korea 2015 CHALLENGES IN PROCESS DEVELOPMENT, SCALE UP & MANUFACTURING OF LOW VOLUME HIGH VALUE BIOLOGICS

Biologics World Korea 2015

CHALLENGES IN PROCESS DEVELOPMENT, SCALE

UP & MANUFACTURING OF LOW VOLUME HIGH

VALUE BIOLOGICS MOLECULES: Global and Indian

Prospect

Dr. Sumant Chaubey, Ph.D

Chief Operating Officer &

Chief Scientific Officer

Bills Biotech Pvt Ltd,

Vadodara,Gujarat India


Global Therapeutic Overview

0%

2%

4%

6%

8%

10%

12%

-10% -5% 0% 5% 10%

Anti-hyperlipidaemics

Anti-bacterials

Bronchodilators

Anti-hypertensive

Oncology

Anti-diabetics Anti-rheumatics

Anti-virals

Bone-calcium regulators

vaccines

Key Drivers

Avastin, Rituxan (Roche),

Erbitux (BMS/LLY/ Merck KgaA)

Glivec (NVS).

Patent Expiries

Taxotere (SAN), Eloxatin (SAN),

Arimidex (AZN), Gemzar (LLY)

Key Drivers

HPV vaccines (GSK & MRK),

Pneumo. (WYE,NVS & GSK) &

Herpes zoster vaccine (MRK).

Plus strong influenza sales.

Key Patent Expiry

Angiotensin II antagonists segment

Diovan (NVS) in 2012, Cozaar (MRK)

2010, Avapro (BMY/ SAN) in 2012

Key Patent Expiry

Lipitor (PFE) in 2011

% Sales Growth: CAGR 2009-15

WW

Mark

et

Share

%

Bubble = WW Sales

$70bn

$29bn

$23bn $26bn

$15bn $15bn

$28bn

$37bn

$34bn

Analysis of Top 10 Therapy Areas in 2014, Market Share & Sales Growth (2009-15)

Oncology potentially outperforming market by both volume & value growth in the space


Global Market : Size & Potential

Ma

rke

t P

ote

ntia

l

In the worst-case scenario, with an estimated price erosion of biologics of up to 30% , potential market worth ~$40bn remains very

appealing & attractive spread across next five years

Year Estimated Revenue of Patented

Drugs Only Biologics

2010 $23bn $2bn

2011 $36bn $1bn

2012 $44bn $8bn

2013 $22bn $17bn

2014 $14bn $9bn

2015 $16bn $13bn

Total $155 $50bn

2010-2015 : Golden period of Biosimilars space to derive advantages from patent expiry

Mc Kinsey report

As per the latest report, Mc Kinsey & Co. expects the average patent expiry at $39.6bn per annum between 2010-2015 as

compared to just $14.2bn in last decade and $16.5bn per annum between 2016-2020. This indicates that 2010-2015 is a

golden period for Global Biosimilars space

Source : US FDA website, Bloomberg, HDFC Securities Institutional Research


Biosimilars Development: SWOT Analysis

Based upon India Brand Equity Foundation , www.ibef.org

The Biosimilars industry is fast-growing and has a strong economic value proposition . However, there are a number of competitive threats that make a well-developed strategy critical to any company wishing to develop in this sector

Weaknesses Cost of Biosimilars products to consumers in emerging markets is still relatively high unlike small molecules generics . Extensive funding is required due to emerging rigorous regulatory requirements.

Lack of widespread awareness and credibility of industry.

Strengths

Lower price point and similar effectiveness to originator

products.

Shorter time to market than originator products.

Higher probability of Return on Investment (ROI) than

with new product R&D.

Due to rapidly increasing healthcare costs, there is high

consumer demand for discounted high quality treatments

Opportunities Large and growing market for biosimilar products. Emerging regulatory frameworks provide structured approval guidelines. High-revenue bio-pharmaceutical projects that have less equivalent Biosimilar approved/available in their portfolio

Threats Future regulations for Biosimilars is still being defined Particularly in US ,few Biosimilars have been formally approved , resulting in little precedence for future rulings. The industry will require greater focus on new investments for future growth


1. The Indian biosimilars (excluding vaccine) market in 2008 was around

about $ 200 million, $ 700 million in 2010 and growing at 30% CAGR.

1. The Indian biologics market consists primarily of vaccines, monoclonal

antibodies, recombinant proteins and diagnostics. 5. The main players of biosimilars in India :

Dr. Reddy’s Ltd. Biocon Ltd. Zydus Cadila Cipla Ltd. Intas Biopharmaceuticals Ltd. and Wockhardt Ltd. .

Biosimilars –Indian Positioning


Opportunity

Scope and Feasibility

Challenges

Mitigations

BIOLOGICS & BIOSIMILAR Manufacturing


BIOSIMILAR DEVELOPMENT @ INDIA: SAVE TIME & COST


What makes Biologics Special?

Biologics have Expected

• Primary, secondary, tertiary,

quaternary structure

• Size

• Charge

• Hydrophobicity

• Folding (S-S bonds)

• Glycosylation

• Bioactivity

• Heterogeneity

& Unwanted:

• Aggregation

• Incorrect folding

• Truncation

• Amino acid modifications oxidation, deamidation,

glycation, etc.


Systematic Approach for Biologics/Biosimilar

development programme

Pre-clinical Phase 1

Phase 2 Phase 3

Phase Development

1. How much test method validation,

product characterization, stability?

2. How tight should specs be?

3. Do I need a bioassay?

Unambiguous Requirements

1.Validated Test Methods

2.Complete Product Characterization

3.Final Specifications

4.Expiry Date Assigned

5.Bioassay Related to Function

6.Full Change Control Program


Biosimilar Product characterizations

• Inadequate characterization data e.g.

– Identity*, heterogeneity/variants (size, charge, hydrophobicity,

glycosylation), aggregates, etc.

– Process-related impurities (HCPs, DNA, antibiotics, chemicals)

• Specifications inadequate (to control quality)

• Note : What to do - Evaluate product as much as feasible before

starting preclinical and clinical studies


Methods for Product Release

• Assay Methods not suitable for intended

purpose

– SEC for Aggregates

– Potency Assay


BioActivity Assays?

• Absence of bioactivity/or Potency assay

specification – Critical quality attribute for proteins

– Proteins inactivated by various conditions

– Potency assay required to evaluate and control product

quality

– Inability to assure consistent dosing of product between

lots; safe dose

• What to do? – Develop and implement a relevant & quantitative

bioactivity and or potency assay and set a

meaningful specification


Viral Safety?

– Cell banks or animal-derived raw materials not

appropriately tested for endogenous or

adventitious agents (mostly viruses, retroviruses)

– Manufacturing scheme should be validated for its

ability to remove or inactivate retroviruses

• Transmission of infectious viruses to humans

• No information on country of origin of ruminant derived

materials used in manufacturing

– Concern over TSEs; BSE


Case Study


Exendin-4 :

1. It enhances Glucose-dependent insulin secretion by

pancreatic beta-cell.

2. Suppresses inappropriately elevated glucagon

secretion.

3. Slows gastric emptying.

Because of this favorable spectrum of Anti Diabetic actions,

Exendin-4 has been widely explored as a potential therapy for

T2DM


Exenedin-4 is a 39 Amino Acid Incretin mimetic

peptide like GLP-1.

Commercially known as BYETTA.

Indicated to improve the Glycemic control in

patents with Type II Diabetes.


Upstream process

Fed-Batch mode

Modified LB medium

Temp. controlled at 35-37C

PH controlled at around 7.0

Agitation 300-500 rpm

Induction with 1mM IPTG

Harvested 4hrs after induction


Production Process

Cell lysis and Triton solubilization.

The fusion protein was purified by GST-Affinity chromatography.

The fusion protein was cleaved by Factor Xa and the peptide was separated by reverse phase and ion exchange chromatography.


Characterization of Recombinant Exendin-4

for its Purity and Activity:


Purity of Exendin-4 by HPLC


Characterization of Exendin-4 by

Mass spectroscopy

Mass Spectrometry Results for rhExendin-4


The receptor activation studies were performed using

RINm5F cells.

Activation of GLP-1 receptor was measured by

quantification of intracellular cAMP after cell lysis.

Bio Assay in comparison with Innovators product.

peptide conc(pM)

1 10 100 1000 10000

cA

MP

rele

ase (

n-f

old

over

basal)

1.0

1.5

2.0

2.5

3.0

VB-63

Innovators Product


tPA Project

To develop process for production of biosimilar version of tPA (alteplase)

√ Cloning of codon-optimized tPA sequence in proprietary vector

√ Create stable CHO clone expressing tPA

√ Create MCB

– Develop purification process (in-process)

– Develop analytical methods (in-process)

– Scale-up process to 3 Liter Bioreactor (in-process)


Cloning of tPA

• Clone tPA expression construct in proprietary

vector

– tPA sequence codon optimized

– tPA subcloned into CHO expression vector

– Confirmed by

• Restriction digestion

• DNA sequencing


CHO Cell Line

Grown untransfected CHO cells (serum-free)

MCB preparation of CHO cells

WCB preparation of CHO cells

Characterize MCB of CHO cells

Sterility

Thaw viability

Mycoplasma

Endotoxins


tPA: Creation of MCB

Expand top clones and prepare R&D cell banks

Expand highest expressing clone and make MCB

Expand single vial from MCB to make WCB (in-progress) Determine antibiotic

sensitivity of CHO cells (kill-curve).

Transfection of tPA expression cassette into suspension CHO cells (serum-free,

using both Turbofect and electroporation)

Selection of transfected cells using Hygromycin

Isolation of polyclones expressing highest activity of tPA

Single cell cloning of polyclones to obtain highest expressing clone

Banked these cells as R&D cell bank


tPA: Purification Process Development

• Approach using clarified cell culture broth

– Capture step- Strong or weak Cation Exchange

– Intermediate purification- Anion Exchange or Hydrophobic

Interaction

– Polishing Step- Cation or Anion Exchange (polishing)

– Affinity step may also be evaluated (need E. coli expression of

DE-3 from Erythrina latissima seeds


tPA: Develop Analytical Methods

tPA Activity- developed chromogenic assay

Estimation of protein content (using Ex. Coeff of 1.9)

Purity- developed SDS-PAGE

Physical appearance and pH test

Estimation of moisture content

Sterility and Endotoxin

In Progress-

• Identification of Alteplase- Tryptic peptide mapping

• Purity- SEC-HPLC (for monomer and single chain content)

• Western blotting of Alteplase

• Fibrinolytic activity assay


MCB & WCB

Lysis & IB Washing 1,2,3.

Solubulization

Refolding And Clarification of G-CSF sample

Ultrafiltration with 30Kd Cassettes And Diafiltration

Ion Exchange Chromatography

Final Bulk

Purity check By SDS PAGE & RP HPLC

Production and Purification process-

GCSF

Fermentation – 15 L


Physical and Biochemical Characterization of rh- G-CSF

• Amino acid sequence of rh G-CSF is compared with the rh G-CSF Data Bank.

Amino acid sequence by MS MS

• Molecular mass scanning over 10,000-35,000 m/z was carried out using sinapinic acid as matrix.

• Mass calibration was done with Apomyoglobulin protein standard as specified by the instrument manufacturer.

MAL DI

• SDS-PAGE analysis was done by using 13.5% Acrylamide gels SDS-PAGE

• RP-HPLC was conducted for purify rh G-CSF and shown to be grater than 98% purity. RP-HPLC

• Different concentrations of WHO G-CSF standard and in house samples were prepared in assay.

BIO ASSAY


Scale-UP Criteria Used

Different scale-up criteria have been used depending on the type of fermentation and the objective of optimization.

The first assumption is geometric similarity between bioreactor vessels of different sizes .

For column purification,hieght of column should kept constant and increase the diameter of column is approach advisable for scale up

However, in some scale-up cases geometric similarity is not preserved. This makes scale-up much more complex.


• Growing mammalian cells in fermenters to produce the protein of interest is a very delicate process. Process parameters like pH or dissolved oxygen concentration need to be controlled very strictly to guarantee the consistency of a product.

• Minor deviations of the predefined process parameters can easily result in changes of product quality attributes like glycosylation, aggregation, c-terminal clipping or acidic variation, which can affect the pharmacokinetics of the protein.


During scale-up, what are the objective parameter, need to optimize (maximize)

product or biomass

cell number/ concentration

product concentration

product activity

volumetric bioreactor productivity


Volumetric Mass Transfer Coefficient, Kla

(KLa)1 = (KLa)2

Where: 1 = small scale bioreactor

2 = large scale bioreactor

criterion is usually applied to aerobic systems

where oxygen concentration is most important and

affects metabolism of the microbial cell.


Volumetric scale-up ratio = V2/V1

= 10,000/80 = 125

Impeller diameter scale-up ratio = Di2/Di1

= 5

SCALE-UP

80 L 10,000 L

i1N

Ni2


Computational Fluid Dynamics (CFD) model from the geometry of a vessel to its oxygen distribution

Characterization of the chosen bioreactors via kL a measurements, mixing time analysis

and analysis for CO2 removal can be performed and compared with the computational

model. This comparison used to validate the applicability of the CFD simulations.

http://www.morbidelli-group.ethz.ch/research/PRP/BenjaminNeunstoecklin/CFD.png?hires





REGULATORY GUIDELINES

FOR BIOLOGICS/BIOSIMILAR

DEVELOPMENT


Design of the Manufacturing Process

“The extent of purification of recombinant DNA products should be consistent with the intended use of the product. Drugs and biologics which are to be administered repeatedly or at high concentrations should be adequately pure to prevent the development of undesired immune or toxic reaction to contaminants.

The purification process should be designed to specifically eliminate detectable viruses, microbial and nucleic acid contamination and undesirable antigenic materials.”

1985 FDA Guidance: Production and Testing

of Recombinant DNA-derived Products


Pharmaceutical Process-Related Impurities Major Safety

Concern for the FDA!

The FDA can place your clinical study in ‘clinical hold’ for the

following CMC reason:

MAPP 6030.1 – “if there are any reasons to believe the

manufacturing or controls for the clinical trial product present

unreasonable health risks to the subjects … such as a product with

an impurity profile indicative of a potential health hazard or an

impurity profile insufficiently defined to assess a potential health

hazard”


Impurity Safety Assessment For Biologics Product-

Related Impurities

Protein Aggregation;

Known immunogenecity

Amino Acid Changes

Immunogenecity (e.g., oxidation of methionine)

Glycosylation Changes

immunogenecity


FDA Guidance For Industry CMC IND Content For

Phase 2 and 3 1999

“Impurities should be identified, qualified, and quantified, as

appropriate. Suitable limits based on manufacturing experience

should be established.”

“For peptides and proteins, characterization should include

data on the amino acid sequence, peptide map, post-transitional

modifications (e.g., glycosylation, gamma carboxylation), and

secondary and tertiary structure information, if known.”


List All Actual/Potential Impurities!

• Process-related impurities

Cell-substrate (DNA, HCP, proteases, endotoxins)

Cell-culture (cell-substrate [DNA, HCP, protease]; endotoxin; media components – antibiotics [tetracycline, gentamicin], hormones [insulin, IGF-1, transferrin], serum)

Purification (enzymes [DNase/RNase]; resin leachates; surfactants; residual cleaning agents]

– Product-related impurities

FDA Guidance for Review Staff and Sponsors: 2004


Identify the “Critical Impurities

‘Critical Impurity’: That which must be controlled to a defined

level to assure appropriate quality and/or safety!

Impurity that impacts patient safety risk

Impurity that is difficult to consistently remove

Impurity that varies from batch-to-batch or changes with

time(case by case)

Risk Management : How you will demonstrate to regulatory agencies adequate control of the specified impurities! Impurity Control Mechanisms

Process validation In-process action limit monitoring End product specification release/stability testing Combination of above


Specifications Through Development

• Specifications Are Expected to Change

– Changes to Analytical Methods

– Evaluation of Stability Data

– Changes to Formulation

– Process Changes and Process Capability

– Enhanced Understanding (Characterization)

– Increased Manufacturing Experience

– Process Validation and Clearance Studies


Assay Methods for Product Release

• Assay methods not suitable for intended purpose

Examples:

– SEC for aggregates: Sample treated to reduce aggregates

before running column

– SDS-PAGE gels under-loaded

– Potency assay: “what is active”

•What are some of the major challenges that are faced in meeting

CMC filing requirements?

•What are the some strategies that are applied to address the

major challenges?


COMPARISON OF NONCLINICAL &

CLINICAL LOTS?

– Product used in animal toxicology studies not

comparable to product intended for clinical

• Can’t rely on Tox studies establishing safety (special

emphasis on impurities, degradants, aggregates)

– What to do?

• Do key Tox studies on appropriate material;

• Do side-by-side comparisons of non-clinical and clinical lots

• Evaluate potential safety impact of differences between Tox

and clinical lots


Stability Testing?

– No stability data or testing plan

• Product stable throughout nonclinical studies

• Product will be stable for duration of clinical studies

– Stable under conditions of use (diluted, etc.)

• Product changes that would result in safety risk

– e.g., release of untargeted toxin

– e.g., release of radiolabel

– e.g., aggregation

– e.g., loss of sterility


Thank You to IMAPAC and associates for Inviting at World

Biologics Korea -2015

Thank You to All Industrial and Academic Researchers and

Colleagues

Documents

CHALLENGES IN PROCESS DEVELOPMENT, SCALE UP ... · PDF fileBiologics World Korea 2015 CHALLENGES IN PROCESS DEVELOPMENT, SCALE UP & MANUFACTURING OF LOW VOLUME HIGH VALUE BIOLOGICS