BIOLOGICALS BIOSIMILARS INTRODUCTION - … set of methods for isolation, ... Prokaryotic cell Eukaryotic cell DIRSCH 10 ... or cell line, in which that DNA is expressed and translated

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BIOLOGICALS – BIOSIMILARS INTRODUCTION

DIRSCH 1

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Natural products

Biologicals

Phytopharmaceuticals

O

O O H

O

O O C O O H

S

N

C H 3

C H 3 C

O

N H C H 2

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PHARMAGOGNOSY

2

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• Biotechnology Production or modification of chemical compounds using organisms or parts of organisms in the context of industrial processes and use. (e.g. bread, Sauerkraut, cheese, beer, ....... vitamins, antibiotics, taxol etc. Pharmaceutical biotechnology)

• Genetic engineering / recombinant DNA technology

A set of methods for isolation, characterisation, amplification and recombination of DNA within and across species boundaries.

• Gene therapy

Transfer of genetic material to tissue cells in order to replace, switch off or complement defective genes.

RELEVANT DEFINITIONS

DIRSCH 3

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LITERATURE used (most in German) Dingermann – Winckler – Zündorf: Gentechnik – Biotechnik Dingermann – Zündorf - Hänsel: Pharmazeutische Biologie Müller-Esterl: Biochemie Löffler – Petrides: Biochemie & Pathobiochemie Alberts: Molekularbiologie der Zelle Web: http://www.deutscher-apotheker-verlag.de/DAZ/

http://www.rote-liste.de/online http://www.ema.europa.eu/ http://www.vfa.de/gentech http:/www.vfa-bio.de/biosimilars

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RECOMBINANT PROTEINS – the principle of production: heterologous expression

Target organism

Cell containing gene of interest

information unit

genetically

modified

organism

"genetic engineering"

Control regions

Extraction and

purification

© Theo Dingermann DIRSCH Host-vector system 5

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Expression vector

Restriction digestion

DNA sequence encoding requested gene product

Recombinant Expression vector

Transformation and propagation of E. coli

Isolation of expressed protein DIRSCH 6

Cloning site DNA sequence encoding requested gene product

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Restriction enzyme or restriction endonuclease

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s

Recognition sequences

sticky ends

DNA fragments

blunt ends

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DNA ligase

8

Sticky ends after EcoR I Restriction

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intergenic sequences

protein-coding genes

non protein- coding genes

Transcripts with unknown function (ca. 21%)

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Eukaryotic cell Prokaryotic cell

DIRSCH 10

Nucleus

Addition of 5‘cap and 3‘Poly(A)tail

RNA splicing

primary DNA transcript

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Posttranslational modifications • Glycosylation • Disulfide bridges

• Cutting off signal peptides/sequences protein synthesis

Ribosome

Golgi apparatus

Endoplasmatic reticulum (ER)

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Since eucaryotic mRNA has a poly-(A)-tail,

complementary oligo-d(T)n is an often used

primer. The enzyme reverse transcriptase (RT)

synthesizes from the 3‘- end of the mRNA a

cDNA copy: with ist endogenous Rnase activity

RT cuts mRNA into pieces. Starting with the 3‘-

end of the cDNA RT degrades mRNA with ist 5'-

3'-exonuclease activity and at the same time

replaces it by complementary DNA. After

ligation of linker DNA and restriction, cDNA can

be inserted into a suitabel DNA vector.

Synthesis of cDNA

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Cloning of DNA

A) Cloning vector

B) PCR

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Multiplication/Colony

Plasmid isolation

Transformation in bacteria

restriciton site

known DNA sequences

DNA sequence of interest

Denaturation at 95°C

Primer hybridisation at 55°C

Polymerisation by Taq-Polymerase

Recombined plasmid vector

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© http://newscenter.lbl.gov/news-

releases/2011/11/29/e-coli-make-three-fuels/ ©http://microbewiki.kenyon.edu/index.php/Sa

ccharomyces_cerevisiae_NEU2011

©http://en.m.wikipedia.org/wiki/File:Cho_cells

_adherend2.jpg ©https://www.dsmz.de/de/downloads/bilde

r/plant-cell-lines.html

Bacteria Mammals /

Mammalian cells Plants / plant cells Yeast

RECOMBINANT PROTEINS – host organisms / expression systems

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Bacterial cell, e.g. E. coli

© WVG Stuttgart: Dingermann/Winckler/Zündorf: Gentechnik Biotechnik, 2011

transgenic bacterium

gene expression

recombinant protein in the periplasm

recombinant protein in inclusion bodies

isolation of recombinant protein

Cell lysis and isolation of inclusion bodies with the denatured protein

Osmotic destruction of the outer membrane and isolation of the native protein

Pros

• Non pathogenic • Entire genome has been sequenced • Extensive experience with bacterial genetics • Easily takes up DNA • Rapid growth • Easy handling • Inexpensive media • Often extremely high product yield

Cons

• No RNA processing • No posttranslational modifications • Proteins often in incorrect tertiary structure • Proteins often denatured („inclusion

bodies“) • Release of endotoxins (pyrogens)

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Pros

• Eucaryot • Rapid growth • Extensive experience with yeast genetics • Entire genome has been sequenced • Cells and their products are non pathogenic • No production of pyrogenic substances • Proteins are usually correctly folded • Often high product yield • Secretion into the culture medium • Some posttranslation modifications possible • Genetically recognized as safe (GRAS) • Easily takes up DNA • Easy handling • Inexpensive media

Cons

• Glycosylations differ largely from mammalian cells (Man6-9GlcNAc2; Hypermannosylation: ManxGlcNAc2)

• Few dominant markers, instead auxotrophy marker

DIRSCH

Baker‘s Yeasts, e.g. Saccharomyces cerevisiae


Nucleus

Transcription

Rough ER

Golgi apparatus

Secretion into culture medium

Signal peptide

Glucan cell wall

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ER (human, yeast)

Golgi (human) Golgi (yeast)

adapted from © Michaela Bogner: Hefe-basierte Produktion von Antikörpern mit verbesserten Eigenschaften, 2010

Complex saccharide structure and terminal NANA

sugar residues of the „High Mannose-Type“

β-1,N-GlcNAc

β-1,4-GlcNAc

β-1,2-GlcNAc

β-1,4-Man

α-1,6-Man

α-1,2-Man

α-1,3-Man

β-1,4-Gal

α-1,3-NANA/α-2,6-NANA

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PROTEIN GLYCOSYLATION affects …

• Charge

• Stability

• Solubility

• Folding

• Cell cell interaction

• Ligand recognition/binding

• Biological function

• Immunogenicity DIRSCH

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Mammalian cells, e.g. CHO cells

© Springer: Dingermann, Hänsel, Zündorf: Pharmazeutische Biologie, 2002 © WVG Stuttgart: Dingermann/Winckler/Zündorf: Gentechnik Biotechnik, 2011

Fixed bed reactor

pump

beads with adherent cells

https://www.youtube.com/watch?v=aK9sNe8c48U

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Pros Cons

• Close relation to „human system“ • Correct folding, even of complex proteins • Secretion into the culture medium • (Almost) authentic posttranslational

modifications

• High production costs (expensive media) • Slow growth • Relatively low protein yield • High investment costs for fermentation • Use of animal-derived medium components

(„Fetal calf serum“, FCS) • High storage costs for master cell lines • „Upscaling“ challenging • Possibly susceptable to human pathogens

(especially viruses)



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Plants (Plant cells)

http://blogs.nature.com/news/2012/05/first-plant-made-drug-on-the-market.html


Pros

• Extensive experience from traditional plant cultivation

• Self insemination • Regenerative ability • Inexpensive cultivation and storage • Variable „Upscaling“ by field cultivation • No human pathogens (especially viruses)

Cons

• Secondary cell wall • Polyploidy • Somaclonal variation • Low product yield • Incorrect glycosylation: simple N-glycans

without sialic acid, unusual links, e.g. 1-3 linked Fucose, Xylose(1-2)Mannose

• „Gene silencing“ • Recombinant protein is generally instable in the

cytosol • Specific regulatory requirements for „release“

of transgenic plants • No GLP/GMP conditions in field cultivation

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Proteins glowing under ultraviolet light on a type of plant used to produce the drug ZMapp. Credit Sean Gallup/Getty Images

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E. coli Yeast

Mammalian cells, such as CHO

complex, glycosylated proteins

„simple proteins“

(amino acid sequence) disulfide bridge

Formation of secondary, tertiary and quaternary structure

modification with sugar residues, lipid chains, sulfate or phosphate residues

© Springer: Dingermann, Hänsel, Zündorf: Pharmazeutische Biologie, 2002

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The European Pharmacopoeia (Ph. Eur.) is Europe’s legal and scientific benchmark for pharmacopoeial standards which contribute to delivering high quality medicines in Europe and beyond. The Ph. Eur. is applicable in 37 European countries and used in over 100 countries worldwide. The European Pharmacopoeia is a single reference work for the quality control of medicines in the signatory states of the Convention on its elaboration. The official standards published within provide a legal and scientific basis for quality control during the development, production and marketing processes. They concern the qualitative and quantitative composition and the tests to be carried out on medicines, on the raw materials used in production of medicines and on the intermediates of synthesis. All producers of medicines and/or substances for pharmaceutical use must therefore apply these quality standards in order to market their products in the signatory states of the Convention.

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EUROPEAN PHARMACOPOEIA 9th edition 2017

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Products of Recombinant DNA Technology

DEFINITION Products of recombinant DNA technology are produced by genetic modification in which DNA coding for the required product is introduced, usually by means of a plasmid or a viral vector, into a suitable micro-organism or cell line, in which that DNA is expressed and translated into protein. The desired product is then recovered by extraction and purification. The cell or micro-organism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the manufacturing process is referred to

as the host-vector system.

This monograph provides general requirements for the development and manufacture of products of recombinant DNA technology. These requirements are not necessarily comprehensive in a given case and requirements complementary or additional to those prescribed in this monograph may be imposed in an individual monograph or by the competent authority. The monograph is not applicable to modified live organisms that are intended to be used directly in man and animals, for example as live vaccines.

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Products of recombinant DNA technology are produced by genetic modification ....

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... in which DNA coding for the required product... ... is introduced, usually by means of a plasmid or a viral vector, ..... ... into a suitable micro-organism or cell line, in which that DNA is expressed and translated into protein. The desired product is then recovered by extraction and purification. .................... host-vector system

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PRODUCTION Production is based on a validated seed-lot system using a host-vector combination that has been shown to be suitable to the satisfaction of the competent authority. The seed-lot system uses a master cell bank and a working cell bank derived from the master seed lot of the host-vector combination. A detailed description of cultivation, extraction and purification steps and a definition of the production batch shall be established. Where products of recombinant DNA technology are manufactured using materials of human or animal origin, the requirements of chapter 5.1.7. Viral safety apply. The determination of the suitability of the host-vector combination and the validation of the seed-lot system include the following elements.

• Cloning and expression • Cell bank system • Validation of the cell banks • Control of the cells • Validation of the production

process

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CLONING AND EXPRESSION The suitability of the host-vector system, particularly as regards microbiological purity, is demonstrated by: Characterisation of the host cell, including source, phenotype and genotype, and of the cell-culture media; Documentation of the strategy for the cloning of the gene and characterisation of the recombinant vector, including: i. the origin and characterisation of the gene; ii. nucleotide-sequence analysis of the cloned gene and the flanking control regions of the

expression vector; the cloned sequences are kept to a minimum and all relevant expressed sequences are clearly identified and confirmed at the RNA level; the DNA sequence of the cloned gene is normally confirmed at the seed-lot stage, up to and beyond the normal level of population doubling for full-scale fermentation; in certain systems, for example, where multiple copies of the gene are inserted into the genome of a continuous cell line, it may be inappropriate to sequence the cloned gene at the production level; under these circumstances, Southern blot analysis of total cellular DNA or sequence analysis of the messenger RNA (mRNA) may be helpful, particular attention being paid to the characterisation of the expressed protein;

iii. the construction, genetics and structure of the complete expression vector. Characterisation of the host-vector system, including: i. mechanism of transfer of the vector into the host cells; ii. copy number, physical state and stability of the vector inside the host cell; iii. measures used to promote and control the expression.

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CELL-BANK SYSTEM

The master cell bank Is a homogeneous suspension of the original cells already transformed by the expression vector containing the desired gene, distributed in equal volumes into individual containers for storage (for example, in liquid nitrogen). In some cases it may be necessary to establish separate master cell banks for the expression vector and the host cells. The working cell bank Is a homogeneous suspension of the cell material derived from the master cell bank(s) at a finite passage level, distributed in equal volumes into individual containers for storage (for example, in liquid nitrogen). In both cell banks, all containers are treated identically during storage and, once removed from storage, the containers are not returned to the cell stock. The cell bank may be used for production at a finite passage level or for continuous-culture production.

Production at a finite passage level This cultivation method is defined by a limited number of passages or population doublings which must not be exceeded during production. The maximum number of cell doublings, or passage levels, during which the manufacturing process routinely meets the criteria described below must be stated.

Continuous-culture production By this cultivation method the number of passages or population doublings is not restricted from the beginning of production. Criteria for the harvesting as well as for the termination of production have to be defined by the manufacturer. Monitoring is necessary throughout the life of the culture; the required frequency and type of monitoring will depend on the nature of the production system and the product. Information is required on the molecular integrity of the gene being expressed and on the phenotypic and genotypic characteristics of the host cell after long-term cultivation. The acceptance of harvests for further processing must be clearly linked to the schedule of monitoring applied and a clear definition of a ‘batch’ of product for further processing is required.

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VALIDATION OF THE CELL BANKS Validation of the cell banks includes: i. stability by measuring viability and the retention of the vector; ii. identity of the cells by phenotypic features; iii. where appropriate, evidence that the cell banks are free from potentially

oncogenic or infective adventitious agents (viral, bacterial, fungal or mycoplasmal); special attention has to be given to viruses that can commonly contaminate the species from which the cell line has been derived; certain cell lines contain endogenous viruses, for example, retroviruses, which may not readily be eliminated; the expression of these organisms, under a variety of conditions known to cause their induction, shall be tested for;

iv. for mammalian cells, details of the tumorigenic potential of the cell bank shall be obtained.

CONTROL OF THE CELLS The origin, form, storage, use and stability at the anticipated rate of use must be documented in full for all cell banks under conditions of storage and recovery. New cell banks must be fully validated.

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© Springer: Dingermann, Hänsel, Zündorf: Pharmazeutische Biologie, 2002

Culture liquid

Tangential Flow Filtration centrifugation (TFF)

Ultrafiltration (UF) Diafiltration (DF)

Anion exchange chromatography

Filtration

UF, DF

Cation exchange chromatography

A

UF, DF

Gel Permeation Chromatography

Bottling, labeling

Product

F

30


VALIDATION OF THE PRODUCTION PROCESS Extraction and purification contaminating substances derived from the host cell or culture medium, including, in particular, virus particles, proteins, nucleic acids and excipients must be removed and/or inactivated Depletion factor, Inactivation factor Characterisation of the final bulk product identity, purity, potency, stability Production consistency

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PROTEIN ANALYTICS: gel filtration/size exclusion chromatography Separation of proteins by size

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PROTEIN ANALYTICS: gel filtration/ size exclusion chromatography Separation of proteins by size

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sample buffer

small protein

large protein

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PROTEIN ANALYTICS: ion exchange chromatography Separation of proteins by charge

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stationary phase

stationary

phase Elution of protein with positive charge

Flo

w d

irec

tio

n

Protein with negative charge binds to stationary phase

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PROTEIN ANALYTICS: affinity chromatography Separation of proteins by specific binding properties

DIRSCH 34

matrix bound transcription faktor

free oligonucleotide

released transcription

factor

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PROTEIN ANALYTICS: electrophoresis analyzes protein

mixtures qualitatively

SDS molecule

Polypeptide chain

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PROTEIN ANALYTICS: polyacrylamide gel Molecular sieve

DIRSCH

Polyacrylamide network

small proteins

sample well

sample

large proteins

Elec

tro

ph

ore

sis

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PROTEIN ANALYTICS: coomassie staining of a SDS gel

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PROTEIN ANALYTICS: isoelectric focusing separates proteins by

differences in their isoelectric point

DIRSCH

negatively charged protein moves towards

the anode

neutral protein diffuses towards the kathode, becomes

negatively charged ... ... and moves back towards

the anode

isoelectric point of the protein

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PROTEIN ANALYTICS: Two-dimensional gel electroporesis

1st dimension

2nd dimension

basic

low molecular weight

high molecular weight

acidic

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SDS Gel with complex

protein mixture

Sandwich with Filter paper

soaked in buffer

Electrophoresis 1. Blocking 2. Primary antibody 3. Secondary antibody 4. Dyes

Dyed protein band

Membrane with transferred

proteins

Other proteins stay invisible

PROTEIN ANALYTICS: Western blot analysis

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colorless, soluble substrates

Secondary antibody conjugated with

alkaline phosphatase

membrane fixed protein

Primary antibody

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PROTEIN ANALYTICS: Enzyme-linked immunosorbent assay (ELISA) quantifies proteins in complex mixtures

Well plate

Specific primary antibody

Other proteins

Analyte

calibration curve

sample absorption

sample concentration

Concentration

Colored product of the enzymatic reaction

Enzyme linked secondary antibody

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BIOSIMILARS – Definition

What are biosimilars? Imitation products of already approved biopharmaceutical medicines which are no longer patent protected. In contrast to generics (imitation products of small molecule drugs), biosimilars are not identical but similar to the original product since the use of living cells / organisms for their production and their structural complexity gives the products a degree of variability which makes the production of identical copies impossible - especially when different production processes are used.

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Generics Biosimilars

Character small molecule drugs „the molecule is the product“

complex molecules, proteins „the process is the product“

Pharmaceutical form predominantly oral Solutions for injection

Production mostly chemical synthesis • Obtained from living cells (complex matrix)

• Highly sensitive processes

Approval • simplified conditions • usually no safety and efficacy

studies required

complex test program, seperately determined for each substance class

Comparison to reference product

Identical active ingredient Proven comparability in quality, safety, efficacy

BIOSIMILARS versus GENERICS

adapted from © Sandoz Biopharmaceuticals 44

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© Dingermann/Zündorf PharmuZ 2012

EXAMPLES of BIOSIMILARS (incomplete list! Snap shot of 2013)

Omnitrope®

Valtropin®

Binocrit®

Epoetin alfa Hexal®

Abseamed®

Retacrit®

Silapo®

Biograstim®

Filgrastim Ratiopharm®

Ratiograstim®

Tevagrastim®

Zarzio®

Filgrastim Hexal®

Nivestim®

Somatropin

Somatropin

Epoetin zeta

Epoetin zeta

Epoetin alfa

Epoetin alfa

Epoetin alfa

Filgrastim

Filgrastim

Filgrastim

Filgrastim

Filgrastim

Filgrastim

Filgrastim

Sandoz

Bio Partners

Sandoz

Hexal

Medice

Hospira

STADA

CT Arzneimittel GmbH

Ratiopharm GmbH

Ratiopharm GmbH

Teva Generics GmbH

Sandoz

Hexal

Hospira

Genotropin®

Humatrope®

Eprex®

Eprex®

Eprex®

Eprex®

Eprex®

Neupogen®

Neupogen®

Neupogen®

Neupogen®

Neupogen®

Neupogen®

Neupogen®

12.04.2006

24.04.2006

28.08.2007

28.08.2007

28.08.2007

18.12.2007

18.12.2007

16.09.2008

16.09.2008

16.09.2008

16.09.2008

06.02.2009

06.02.2009

08.06.2010

Handelsname INN Vertreiber Referenzprodukt Zulassung

Human growth hormone

Erythropoetin

Granulocyte-colony stimulating factor (G-CSF)

Remicade®

Remicade®

Remsima®

Inflectra®

Infliximab

Infliximab

28.06.2013

28.06.2013

Celltrion

Hospira Monoclonal antibodies (against TNF) DIRSCH 45

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BIOSIMILARS – reference products (originator products) also have biological variability:

Schiestl M et al, Nature Biotechnology 29, 310–312 (2011)

Molecular variations (within defined specification limits) also exist between batches. Changes in the production process can cause greater variations, which are reported to the regulatory authority for thorough examination and approval.

The analysis of various batches of rituximab showed different glycosylation patterns (left), depending on the expiry date, and thus different ADCC (antibody dependent cellular cytotoxicity) potencies (right). light blue dots: before dark blue dots: after a change in the manufacturing process

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„Micro-heterogeneity“

Protein drugs, such as insulin or monoclonal antibodies, have molecular weights up to 1000 times larger than „small molecule“ drugs. As the molecular weight increases, the complexity of the molecule and the possibility of product heterogeneity increase.

BIOSIMILARS – complexity and product heterogeneity

© Isam Rais, PharmuZ 2012

complexity

47

synthetic drug biological drug

Monoclonal antibodies 150.000 Da

Acetyl salicylic acid 180 Da

Insulin 5700 Da

Complexity

IgG monoclonal antibody C6466H9982N1726O2024S40

MW = approx. 150.000 Da

N-terminal pyro-glutamate Methionine oxidation Deamidation Excision of C-terminal lysine Glycation Fragmentation Glycosylation

Examples of possible mikroheterogeneities:

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BIOSIMILARS – Analytics

Overview of analytical methods for the characterization of proteins

Analytic principle Methods Application

Electrophoresis SDS-PAGE, Isoelectric Focusing (IEF), Capillary Gel Electrophoresis (CGE)

Purity (aggregates, fragments), charge heterogeneity

Chromatography RP-HPLC, Size Exclusion Chromatography (HP-SEC), Ion Exchange Chromatography (IEC), Hydrophobic Interaction Chromatography (HIC)

Purity, charge heterogeneity

Mass Spectrometry Peptide Mapping Identity, chemical modifications

Spectroscopy Circular Dichroism (CD), Differential Scanning Calorimetry (DSC)

Spatial structure

Bioassay, binding assay Cell based assay formats, Biacore, ELISA Identity, potency

adapted from © Isam Rais, PharmuZ 2012 48

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DIRSCH adapted from © Isam Rais, PharmuZ 2012

BIOSIMILARS – Development

The development of biosimilars is a is a step by step process. By analysis of originator batches, a Target Product Profile which serves as a target for process development can be created. On the basis of this TPP, a manufacturing process for a drug with similar properties is developed. Subsequently, the new drug is compared with the originator product in detailed studies.

development of a manufacturing process

Similarity/ comparability exercise

Characterization of the reference product batch

Definition of a target product profile (TPP)

Implementation of Quality by design (QbD)

Optimization of upstream and downstream

processes

Defined product profile of the target structure

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BIOSIMILARS – Development – comparability exercise

Reference product (Biopharmaceutical comparator product)

Physicochemical and biological

characterization

Non-clinical PK/PD/safety

studies Clinical studies Quality

Structure of the biosimilar,

manufacturing process, ...

adapted from © Isam Rais, PharmuZ 2012

Each aspect of the product development must always be carried out in direct comparison to an approved reference product. Comparison with literature data alone is not acceptable.

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DIRSCH adapted from © Isam Rais, PharmuZ 2012

Physicochemical characterization

Biological characterization

Preclinical studies

PK/PD studies in humans

Clinical studies

Sim

ilari

ty

Co

mp

arab

ility

Val

idat

ion

BIOSIMILARS – Development – comparability exercise

A step by step approach is necessary in order to achieve the label „Similarity“. It starts with physicochemical „comparability“ and, as the amount of acquired data increases, the highest step of „Similarity“ is reached.

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BIOSIMILARS – Regulatory approval

adapted from © Isam Rais, PharmuZ 2012

Extent and proportion of reporting and documentation requirements for a biosimilar in comparison to a new drug

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BIOLOGICALS – BOSIMILARS – BIOIDENTICALS – BIOBETTERS

Dingermann & Zündorf: Biobetter – Kopien besser als das Original, PZ 06/2015: http://www.pharmazeutische-zeitung.de/index.php?id=56229

Biologicals – 1st and 2nd generation drugs Bioidenticals – Biologicals with different names that are actually identical because they were manufactured in the very same process Biosimilars – generic versions of a recombinant drug that are the closest possible replica of their originals. Ultimately, they are similar but not identical to the original. Biobetters (Biosuperiors) – copies or improved versions of a biological drug (for example with modified pharmacokinetic or -dynamic properties); see also „muteins“

2nd generation

drugs

identical to human structure

1st generation

drugs

technical limitations

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Documents

BIOLOGICALS BIOSIMILARS INTRODUCTION - … set of methods for isolation, ... Prokaryotic cell Eukaryotic cell DIRSCH 10 ... or cell line, in which that DNA is expressed and translated