developing a biotherapeutic from clone to clinic.ppt · biotechnology industry organization, 1990....

Developing a Biotherapeutic:From Clone to Clinic®From Clone to Clinic®

Sheila G. Magil, PhDBi P T h l C lt t IBioProcess Technology Consultants, Inc.

Advances in Chemical Sciences“Bench to Pilot Plant” Symposium

Cambridge, MA23 October 2009

Outline of PresentationIntroduction

A Biologic is just like a NCE…except when it isn’t

Development Overview

Special Points

Summary

From Clone to Commercial®

A biologic is just like a NCEExcept when it isn’t

• Size

• Complexity

Old vs New Definition

• Process is the product

• Well‐characterized

What does that mean?


Biologics and Biopharmaceuticals

Broad Definition of Biologics Products

d d b C d f• Products Made by or Composed of Viable Organisms and Biopolymer Analogs

Natural & rDNA ProteinsNatural & rDNA Proteins

Monoclonal & Polyclonal (natural) Antibodies

Hormones, PeptidesHormones, Peptides

Antibiotics, Plant & Animal Extracts, Allergens

Vaccines, Cell & Gene Therapyacc es, Ce & Ge e e apy

Human & Xenogenic Cells & Tissues

Blood & Blood Derivatives


Biologics Have Multiple Uses

Source: Source: Adapted from: BIO. "Biotechnology in Perspective." Washington, D.C.: Adapted from: BIO. "Biotechnology in Perspective." Washington, D.C.: Biotechnology Industry Organization, 1990.Biotechnology Industry Organization, 1990.

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Relative Size of a Biotherapeutic and a Chemical Drug

From: Behram, Rachel E. (2008, November 21) Follow-on Biologics: A Brief Overview.Presentation given by the U.S. FDA at the U S F d l T d C i i k hU.S. Federal Trade Commission workshop

"Competition Issues Involving Follow-on Biologic Drugs."


Complexity Example Antibody Structure


From Kozlowski and Swann

Biologics vs. Drug Development: Mean Clinical Development and Approval TimeBiologics vs. Drug Development: Mean Clinical Development and Approval Time

ears

Ye

From Clone to Commercial®Reference: DiMasi, 2003

FDA and BiologicsOver 200 Years of Regulation• Prehistoric Era

1798 – Marine Hospital Service (PHS)1800s – Vaccination & Antitoxins1901 – St. Louis Diphtheria Antitoxin Deaths1902 – Biologics Control Act, ELA/PLA Procedure

• Pre‐FDA ERA 1902‐72

PHS/NIH, But with FDA Regulation of Antibiotics, Plant/Animal Isolates, Insulin/Hormones

• FDA Early Era 1972 80’s• FDA Early Era 1972‐80’s• FDA Modern Era 1980’s‐96

1988 – CBER Created1st Biotech products – rhGH, rhInsulin, OTK3, α‐IFN1 Biotech products rhGH, rhInsulin, OTK3, α IFN

• FDAMA Era Post‐97

Elimination of ELA“Well Characterized Biologics” & “Comparability Protocols”


Traditional Biopharmaceuticals

Traditional Biologics Defined by Manufacturing Process.

Manufacturer Had to Directly Control & Own Entire Process• Contents were Undefinable (Blood & Vaccines)

• Difficult to Demonstrate Purity, Identity, Contamination, Potency & Consistency

Early Recombinant & Monoclonal BiologicsWeren’t Well Characterized.

Because of the limited ability to characterize & control identity and structure, and measure the activity of clinically‐active components, a biologic was defined by its manufacturing process.process.

FDA presumed that changes in the manufacturing process, materials, equipment or facilities changed the biological product.

FDA expected manufacturers to make manufacturing changes during early development so that the changes could be validated during the product’s pivotal clinical trials.

Process Essentially Frozen by Phase I/II


Current BiopharmaceuticalsMore complex with significant tertiary structure and post‐translational modifications

Many current biologics can be manufactured, characterized and assayed with drug‐like control and resolution

Can be Manufactured, Characterized and AssayedWith Drug‐Like Control & Resolution

• Tightly Defined Production Processesg y

• High‐Resolution Analytics

• Significant Understanding of Structure/Composition‐Activity Relationships

Evolved Regulatory Policies toMeet Needs for Dominant Class of Agents

• Greater FDA Understanding of Technology

• cGMP in the 21st Century Initiative

• Risk‐based decisions


FDA & Biopharmaceuticals Now

CBER RegulatesMonoclonalsMost rDNA proteins

CDRH RegulatesFewEngineered Tissues

CDER RegulatesMany

Non‐Blood/Immune N l P iBlood & Blood Derivatives /

AnalogsVaccines, Allergens Viable Agents (C&GT)

Some HemostaticsOrthopedic ImplantsIVDs

Natural Proteins & rDNA Products

• Hormones, Aprotinin, Heparin, Insulin, hGH

M A i & S h iViable Agents (C&GT)• Microbial/Plasmid• Cellular• Xenogenic

Bl d B k Di ti

CVM Regulates Biotech Animal Drugs & Most Biologics

• Most Anti‐sense & Synthetic Peptides

• Biological Sourced “Small Molecule” A tibi tiBlood Bank Diagnostics

Miscellaneous CFSAN Regulates Biotech Food & Food Additives

−Antibiotics−Oncology− Sterols

Miscellaneous


Overview of Development of a BiologicProduct Concept

Process DevelopmentProcess Development

Analytical Development

Preclinical StudiesPreclinical Studies

Clinical Studies

Scale up

Validation

Filing and Approval


General Scheme for Biomanufacturing

Drug Substance (API)Drug Substance (API)Expand CultureExpand Culture

Working Cell Bank (WCB)Working Cell Bank (WCB)

InnoculumInnoculum PreparationPreparation

Production BioreactorProduction BioreactorFormulationFormulation (optional)(optional)

Drug Substance (API)Drug Substance (API)Expand CultureExpand Culture

Maximize Cell Density and Product Maximize Cell Density and Product ExpressionExpression

Aseptic ProcessingAseptic Processing

Primary RecoveryPrimary RecoverySeparate Cells, Concentrate

Stabilize Product

Transfer to Final Drug Product Transfer to Final Drug Product ConditionsConditions

Drug ProductDrug Product

PurificationPurificationRemove Majority of Process and Product

Contaminants and Impurities

Bottle, Lyophilize (if appropriate))

FormulationFormulation

PolishingPolishingRemoval of Trace Impurities


FormulationFormulationTransfer to Drug Substance ConditionsTransfer to Drug Substance Conditions

Development Timeline and Costs


Selecting a Production SourcePotential Sources

• Prokaryotic or Eukaryotic

Bacteria, Fungi, Insect cells, Mammalian cells

TransgenicsTransgenics


Bacteria as a Production SystemAdvantages

• Well understood molecular biology

Disadvantages• Endotoxins• Refolding & separation of gy

• Simple vector construction

• Rapid cell growth

• High intracellular expression

g pincorrectly folded from properly folded product

• Lack post‐translational difi i• High intracellular expression

levels

• Secretion into periplasm possible

Si l ll b k h t i ti

modifications• Micro‐heterogeneity• Poor extracellular expression

F i t b i d• Simple cell bank characterization

• Easy to grow in inexpensive media

E bli h d l k

• Fusion partner may be required• N‐terminal methionine

• Established regulatory track record


Yeast as a Production SystemAdvantages• Lack endotoxins; GRAS

Disadvantages• Heterologous proteins may be

• Large‐scale fermentation technology established

• Low media costs

incorrectly glycosylated and folded

• Recombinant proteins are Low media costs

• Genetics well understood

• Proteins properly folded

generally overglycosylated

• Complex vector construction

• Low intracellular expression• High expression levels & rapid

growth

• Natural secretor

• Low intracellular expression

• Difficult to lyse


Mammalian Cells as a Production SystemAdvantages• Correct post‐translational

Disadvantages• Expensive

modifications

• Properly folded proteins

• Easily secreted

• Require expensive media

• Slow growth & low production levelsEasily secreted

• Good regulatory track record

levels

• Potential oncogene contamination

E i ll b k• Extensive cell bank characterization


Approximate Productivities

Microbial fermentation: 10‐100 g/L –dependent on product formation of inclusion bodies secretion ofproduct, formation of inclusion bodies, secretion of product, etc.

Mammalian cell culture: 0.01‐3 g/L – Mabs at high end (~1 g/L); rProteins lower

Animal transgenics: 2‐20 g/L in milk

Plant transgenics: Expression levels in corn of approximately 0.01 – 0.25% of dry seed weight


pp y y g

Introduction toFermentation and Cell Culture

Fermentation and Harvest

Fermentation Conditions

Product Production

Harvest

Cell Breakage

Inclusion Body Collection

Solubilization

Refolding

Clarification


Clarification

Centrifugation

• Disc‐stack

• Decanter

Depth Filtration

Microfiltration

• Separation of particulates

• Filters rated by pore size

Ultrafiltration

• Separation of macromolecules

• Filters rated by MW cut‐off


Expanded Bed Adsorption

Objectives of Primary Recovery

Product Isolation/Extraction

ClarificationClarification

• Solid‐liquid separation

ConcentrationConcentration

• Removal of main contaminant – water

StabilizationStabili ation

• Remove proteolytic enzymes

Purification (not primary objective)( p y j )

• Remove Bulk of Impurities


Purification and Polishing – Objectives

Purification• Remove bulk of contaminantsRemove bulk of contaminants

HCP, DNA, media components• Remove most product‐related impurities• Concentrate• Stabilize

Polishing• Removal of trace host cell contaminantsRemoval of trace host cell contaminants• Removal of product‐related impurities• Concentrate & Buffer Exchange


Potential Process Impurities & Contaminantsin Biologics

Media Components

• serum proteins

Host Cell Protein

DNAp

• amino acids

• lipids

• steroids

Viruses

Endotoxin

Process Chemicals• vitamins

• sugars

• trace elements

Process Chemicals

Leached Affinity Ligands


Critical Issues for Scale-up and Validation

Establish a causal relationship between process, product structure and product functionproduct structure, and product function

Key to the Quality by Design Approach


Overall Yield vs. Number of Steps

100%

70%80%90%

100%

50%60%70%

95%90%80%ll

Yie

ldll

Yie

ld

20%30%40% 80%

70%

Ove

raO

vera

0%10%

1 2 3 4 5 6 7 8

From Clone to Commercial® Number of StepsNumber of Steps

Cell Culture Optimization

Recombinant Protein ProductionRecombinant Protein Production

Batch Fed-batchFed-batch,Optimized

Yearly production (Kg) 10 10 10

Recovery yield 60% 60% 60%

Expression level (mg/L) 70 225 235 >8gSuccess rate 90% 90% 90%

Bioreactor volume required (L) 264,550 82,305 78,802 2125


Optimization Parameters: Bioreactor

Seed train

• Initial inoculum concentration and preparation• Initial inoculum concentration and preparation

• Optimal dilution for expansion

Media content

• Cell growth and expansion phase

• Production phase

Production parameters

• Mammalian: batch versus perfusion

• Microbial: secreted versus intracellular expression

• Induction of protein expressionInduction of protein expression

• Time of protein production


Optimizing the Refolding Process

Urea vs. Guanidine

• Smaller dilution factor required for refolding• Smaller dilution factor required for refolding

• Less aggregate formation

• Higher recovery• Higher recovery

• More amenable to downstream processing


Comparability…….

“FDA recognized that improvements in production methods, process and control test methods, and test methods for product characterization have allowed manufacturers of biological products to readily identify and assess the impact of changes made to production processes and production facilities. For example, techniques for isolation of macromolecules, product and process related, have improved greatly in recent years. The manufacturer’s ability to establish sensitive and validated assays for characterizing the product and biological activity and to evaluate the significance of differences noted in such assays can provide the basis for FDA to assess product comparability without the necessity of repeating clinical efficacy studies.”

FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, Including Therapeutic BiotechnologyIncluding Therapeutic Biotechnology--Derived Products. CBER/CDER, April 1996.Derived Products. CBER/CDER, April 1996.


Analytical MethodsAnalytical MethodsIn-process, Release and Characterization


Analytical Development

Analytical development concurrent with process development

• In‐process monitoring

• Assays for final product specifications

Quality control assays

• Assess safety and comparability of product from run to run

• Specifications often provided by regulatory agencies

Potency assays

• Required for final product release

• Assay must correlate directly with therapeutic effect

• Refined during pre‐clinical and clinical development


Safety & Effectiveness Factors

Product & Process Design and l i i lControl Are Critical

• Identity, Potency & Purity

I it & I it P fil• Impurity & Impurity ProfileProduct & Process ContaminantsAdventitious Agents & Removal

Subtle ChangesSubtle Changes• Immunogenicity & Antigenicity

• Pharmacokinetics

bili

Subtle ChangesSubtle Changesin Product or in Product or Process Can Process Can

Have Astounding Have Astounding • Stability

• Consistency

ggBiological EffectsBiological Effects


QC Methods

HPLC (RP, SEC, IEC, HIC)

Electrophoretic Methods (SDS and Normal, Reduced and Non‐d d C i d Sil t i IEF W t Bl tti )reduced, Coomassie and Silver stains, IEF, Western Blotting)

ELISA

BioassayBioassay

Microbiological Methods (Bioburden, Sterility, Viability, Microbial contamination)

Endotoxin

Carbohydrate characterization

S i (UV Vi CD FTIR Fl )Spectroscopic (UV‐Vis, CD, FTIR, Fluorescence)

TOC

USP/EP/BP/JP raw material testing


USP/EP/BP/JP raw material testing

Potency Assay

Monoclonal Antibody Potency Assays

• Binding to target (Synagis)• Binding to target (Synagis)

• Complement dependent cytolysis (Campath)

Enzymatic Potency AssaysEnzymatic Potency Assays

• Colorimetric assay correlates with activity

Growth Factor Potency AssaysGrowth Factor Potency Assays

• Cell growth in response to product

• Activation of known responsive signalp g


Analytical Methods and the Information Providedfrom an FDA Presentation by A. Mire-Sluis

Method Size Charge Structure Purity PotencyMS +++++ + 1° +++++ +++++ -

CD - - 2°/ 3°/ 4°++++ ++ -

NMR + - 1°/ 2°/ 3°++++ ++ -

HPLC: SE +++ - 2°/ 3°/ 4°+++ +++ -

HPLC: IE - ++++++ 1° +++ +++ -

HPLC: RP + + 1°/ 2° +++ ++++ -

Electrophoresis, Reducing

+++ - 1° +++ +++ -

El t h i N 1°/ 2°/ 3°+++ +++Electrophoresis, Non-reducing

- - 1°/ 2°/ 3°+++ +++ -

IEF + ++++ 1°/ 2°/ 3°/ 4°+++ +++ -

Immunoblotting +++ + 1°/ 2°/ 3°/ 4°+++ - -

Immunoassay - - - / + - -

Receptor Binding - - 1°/ 2°/ 3°/ 4°+++ - -/+++

Bioassay - - 1°/ 2°/ 3°/ 4°+++ - /+ +++++


Characterization analyses

Protein Modification “Hot Spots”

• Amino acid substitutions

• Truncated forms (clipped or cleaved)

• Mis‐matched disulfide bonds

• N and C‐terminal heterogeneity

• Aggregation

• Dissociation

• Carbohydrate heterogeneity

• Post translation modifications


CharacterizationPost‐translational modifications

• Acetylation

• Acylation• Acylation

• Addition of lipid

• Amidation and deamidation

• Carbamylation

• Carboxylation

• Formylaton

• γ‐carboxyglutamic acid

• Methylation

• Oxidation

• Phosphorylation

• Sulphation


Viral ClearanceViral Clearance


Virus Clearance Validation/Evaluation/Study

• Viral clearance studies are required for products d i d f li ll l l dderived from mammalian cell culture, plasma, and other sources of potential risk.

• Clearance studies are performed by spiking with model viruses that can be cultivated to a high titer.

Clearance is achieved by inactivation and/or removal.


Methods for Viral Removal/Inactivation

RemovalFiltration

InactivationHeat• Filtration

• Chromatography

• Heat

• Detergent

• Low pH• Low pH

• NaOH treatment


Process Validation . . .

“...has indeed, in some quarters, assumed the status of a new religion, with its own mystique, its own ceremonies and its own special incantations, expressed in an arcane language understood only by initiates.”

J R Sh 1985J.R. Sharp, 1985


Process Validation


Critical Issues for Validation

Upper Upper EdgeEdge--ofof--failurefailure

Upper Upper PARPAR

Upper Upper Control Control Limit Limit

Upper Upper Operating Operating

LimitLimit

Lower Lower Operating Operating

LimitLimit

Lower Lower Control Control LimitLimit

Lower Lower PARPAR

Lower Lower EdgeEdge--ofof--failurefailure

NORNOR

PARPAR

MORMOR

PARPAR

NOR = Normal Operating Range (Batch record)NOR = Normal Operating Range (Batch record)MOR = Manufacturing Operating Range (BLA)MOR = Manufacturing Operating Range (BLA)PAR = Proven Acceptable Range (Validation)PAR = Proven Acceptable Range (Validation)


PAR Proven Acceptable Range (Validation)PAR Proven Acceptable Range (Validation)

Validation: Quality AssuranceValidation is a fundamental part of Quality Assurance

Quality, safety, and effectiveness must be designed and built into the product

Quality cannot be inspected or tested into the finished product

Each step of the manufacturing process must be controlled to maximize the probability that the finished product meets all quality and design specificationsspecifications

“Guideline on General Principles of Process Validation,” May 1983


Cleaning Validation

Validated assays are required

• Specific sensitive• Specific, sensitive

• Broad spectrum

Test methodsTest methods

• Rinse fluids

• Swab testingSwab testing

• Coupons

• Visual inspectionp


SummaryDeveloping a biotherapeutic is just like developing a small molecule drug…except

Increased complexity of molecule

Variability is in drug substance

Analyses of drug requires many orthogonal methods and many more tools

Need to demonstrate removal of advantitious agentsNeed to demonstrate removal of advantitious agents

Cleaning validation


Thank you!BioProcess Technology Consultants, Inc.

289 Great Road, Suite 303

Acton, MA 01720

978‐266‐9110

[email protected]

or

www.bioprocessconsultants.com


developing a biotherapeutic from clone to clinic.ppt · biotechnology industry organization, 1990....

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