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Biological medicinal products

Dr. Herman J. Woerdenbag

Department of Pharmaceu1cal Technology and Biopharmacy
University of Groningen
The Netherlands
h.j.woerdenbag@rug.nl

2016 H.J.Woerdenbag

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Deni9on and domain

Biological medicinal products
Biopharmaceu1cals
Biologicals

Therapeu1c agents prepared by a biological process

Medicines containing ac1ve agents of biological origin

Small molecule drugs (MW <900 Dalton) (mostly organic)

versus biomolecular drugs (biomacromolecules)
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Biological medicinal products

Pep1des and proteins
Vaccines
ATMPs (Advanced Therapy Medicinal Products)
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Major characteris9cs
Composed of proteins, nucleic acids, sugars or (complex)
combina1ons of these
Isolated from human or animal sources or from micro-
organisms
Produced biotechnologically
DNA recombinant techniques
Characterisa1on is complex (dierent from low mol wt API)
Physicochemical tes1ng
Biological tes1ng ac1ve substance and nal medicinal product
Produc1on process and quality control
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Market posi9on
Biopharmaceu1cal medicinal products currently comprise
about 25% of newly approved drugs
Licensed products
Engineered proteins
Not yet worldwide licensed products, but in clinical trials
Gene therapy
Increasingly important
Medical elds
Oncology, rheumatology
Cardiology, gastroenterology, neurology, dermatology
Inherited diseases (deciencies)
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Proteins and pep9des

Amino acids (20, L-form) linked by amide bonds
Pep1des: <~50 amino acids; no ter1ary structure
Amino acid sequence dened by specic nucleo1de base pair
sequence
Post transla1onal modica1on
Glycosyla1on
Protein folding (3D structure)

Transla1on to mRNA
Transcrip1on to protein
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Therapeu9c pep9des and proteins

Pep1des (examples)
Oxytocin
Gonadorelin
Goserelin

Proteins (examples)
Enzymes (pancrealipase)
Monoclonal an1bodies (bevacizumab, trastuzumab)
Cytokines (interferons, interleukins, tumour necrosis factor)
Hormones (insulin, glucagon, human growth hormone)
Hematopoie1c blood factors (erythropoie1n, colon s1mula1ng factor)
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Archetype therapeu9c proteins

Extracted from living organisms (animals, humans)
Insulin from animal (pig) panceas 1ssue
Growth hormone from human cadaver pituitary glands
An1bodies for passive immunisa1on (horse serum)
Vaccines produced in eggs
Ini1ally the only op1on, but
Dicul1es in extrac1on
Very low yields
Immunogenicity (foreign proteins, small dierences in structure)
Danger of transmission of infec1ous diseases
- Creutzfeld-Jacobs: donor pituitary glands infected with prions
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First genera9on
Mainly copies of endogenous (human) proteins or an1bodies
Produced by recombinant DNA technology
Transfec1on of human gene into suitable expression system
- Heterologous produc1on system
Harves1ng and purica1on
First product: insulin
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Second genera9on
Engineered products
Gene altered
End product altered
To improve performance
Pharmacokine1c and pharmacodynamic proper1es
To act longer, faster, bemer

Applica1ons: recombinant hormones, therapeu1c

monoclonal an1bodies
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Engineering proteins
Prior to expression: altera1on of nucleo1de sequence of the
gene coding for the protein
Change single amino acid
Change whole region of polypep1de chain
Why?
Modica1on pharmacokine1c and pharmacodynamic proper1es
Genera1on of novel fusion proteins or other proteins
- Fusion protein: two or more proteins engineered to be expressed as
one single polypep1de chain (may be joined by short linker)
Reducing immunogenicity (humanizing)
Post-transla1onal: PEGyla1on
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Examples 2nd genera9on products

Type of change Protein Indica9on Reason for change
Altered amino acid Insulin Diabetes Faster ac1ng hormone
sequence
Interferon analogue An1viral Superior ac1vity

Tumour necrosis factor Rheumatoid Prolongs half life
receptor human disease
immunoglobulin G Fc
fusion protein
Altered carbohydrate Erythropoie1n Anaemia Prolongs half life
residues analogue
Covalent amachment Interferon Hepa11s C Prolongs half life
to polyethylene glycol
Human growth Acromegaly Prolongs half life
hormone

Source: Rang & Dales Pharmacology

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Third genera9on
Future?
Proteins designed and constructed from scratch
Based on knowledge of biological target
Products not found in nature
Synthe1c biology domain
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Heterologous expression systems (1)

Each system has specic advantages and disadvantages

Bacteria (Escherichia coli)

Fast grow
Easy to manipulate
Bacterial toxins
No post-transla1onal modica1on (no glycosyla1on, needed for ac1on)

Yeast cells (Saccharomyces cereviciae, Pichia pastoris)

Easy to culture
Post-transla1onal modica1on
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Heterologous expression system (2)

Mammalian cells (Chinese Hamster Ovary, CHO)
Less easy to culture (compared to bacteria)
Low yields
Slow growth
Higher costs

Insect cells (bumery, y)

Post-transla1onal glycosyla1on
Osen high yields
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Heterologous expression system (3)

Plants
Nico7ana tabacum L.
Edible plants (banana)

Transgenic camle
Cow: milk (containing therapeu1c protein)
Rabbit: therapeu1c protein bioreactor
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Produc9on
Gene of interest transfected (using plasmid, virus) into host
cell (produc1on system)
Stringent regula1ons
Sterile culture condi1ons, clean rooms, bioreactors
Purica1on by centrifuga1on and/or ltra1on
Characterisa1on and quality control using spectroscopy,
chromatography, calorimetric techniques
Complexity!
Low yields, expensive
Dierent produc1on methods, dierent batches
Comparability and reproducibility?
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Formula9on of protein drugs

Degrada1on occurs easily
Inac1va1on upon storage
Easy and fast in vivo clearance
Dicul1es in reaching therapeu1c target

How to increase stability?

Especially instable in solu1on
How to op1mise bioavailability?
How to improve delivery and targe1ng?
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Stability
Maintenance of stability upon storage
Deamina1on, pep1de bond hydrolysis, oxida1on, Maillard reac1on
Denatura1on (altera1ons in secondary and ter1ary structure)
Hea1ng, freezing, extremes of pH, organic solvents
Protein aggrega1on in solu1on
Non-na1ve aggregates: immune response
Yet invisible aggregates: few molecules

Stability improvement
Lower pH (3-6), freeze drying, oxygen exclusion, chela1ng
agents, stabilisers to prevent aggrega1on (polyols, sugars)
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Ecacy
Degrada1on in gastrointes1nal tract
Poor transport across biological membranes
Parenteral administra1on needed
S1ll rapid clearence
Polyethylene glycol conjugates prolong ac1vity of proteins
- PEGylated
Polymer matrices applied to sustain ac1vity of pep1des

Other routes of administra1on?

Nasal, pulmonary
Op1misa1on of bioavailability
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Biosimilars (1)
Biological medicinal product equivalent to biological
medicinal product that has previously obtained marke1ng
authorisa1on (biological reference medicinal product)
Similar / equivalent but not iden1cal
Cost reduc1on
EU Guideline on similar biological products (23.10.2014)

For comparison: small molecule drugs

Generic drug, iden1cal to branded drug, can be produced and
marketed
Bioequivalence tes1ng
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Biosimilars (2)
Biological medicinal products are complex of nature
Product of other manufacturer may not be exactly iden1cal to that of
original manufacturer (patent holder, produc1on process proprietary)
- Dierent strains, dierences in produc1on process
- Dierent eect possible
Impuri1es may be present
- Safety aspects

Thorough demonstra1on of comparability required

Pa1ent switches from one biological medicinal product to a

biosimilar: careful monitoring (and recording) necessary
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Immunological defense (intermezzo)

Vaccines
An1bodies
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Immune system
Mechanism to combate infec1ons, promote healing and
restora1on
Cellular and molecular guards in the body
Outside barriers: skin, gastrointes1nal mucosa, lungs
Compromised by immunosuppressant drugs
Disorders of immune system: autoimmune diseases,
inammatory diseases, cancer
Two systems, working together with a variety of mediators
and mechanisms
Innate (non-adap1ve)
Acquired (adap1ve)
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Innate immune system

First line of defense
Non-adap1ve, non-specic response
Developed early in evolu1on
Present in most mul1cellular systems
Ac1vated immediately following injury or infec1on
Mediators generated by macrophages, mast and dendri1c cells: cytokines
(interleukin-1), tumor necrosis factor (TNF)-, chemokines
Release of prostaglandin, histamine, leukotriene
Vasodilata1on, oedema
Amac1on of leukocytes, phagocytosis, killing of invador
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Aquired immune system

Second line of defence
Adap1ve reponse, ac1vated by innate response
Boosts eec1veness of innate responses
Appeared much later in evolu1onary terms
Only in invertebrates
Immunological memory
Recogni1on at second exposure, ac1va1on of acquired immune system
More powerful than innate immune system
Highly specic
Lymphocytes (B-cells) are key cells
An1body-mediated component
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An9gen an9bodies
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Vaccines (1)
Administered prophylac1cally
Protec1on against infec1ous diseases
Vaccines: an1gens, which ac1vate acquired immune system
Produc1on of an1bodies against an1gens
Induc1on of immunological memory
Enable immune system to recognise and destroy specic pathogens if
exposed to pathogen the second 1me
Three types
Live organisms, amenuated
Inac1vated (heat, chemical)
Subunit vaccines
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Vaccines (2)
Produc1on
Bacterial vaccines in bioreactors
Viral vaccines in fer1lized chicken eggs (u vaccine)
Recent development: mammalian cells (MDCK)
- Ini1al infec1on mul1ple copies of virus produced which infect other cells
- Harves1ng at 1me that 1tres are suciently high
Recombinant vaccines
- Inser1on of gene coding for an1gen into bacterial, yeast or mammalian cell
- Mul1ple copies are obtained (hepa11s B, human papiloma virus)

Isola1on and purica1on

Ultracentrifuga1on, chromatography
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Vaccines (3)
Stability osen poor
Con1nuous cold chain (costs, logis1cs)
Dehydra1on before use
- Sugar glass technology

Formula1on
Preserva1ve (thiomersal): mul1ple dose vaccines
An1bio1c to prevent bacterial growth
Stabilisers (2-phenoxy esters)
Adjuvants (alumen, imidazoquinolones) (adverse reac1ons)
New developments (administra1on, formula1on)
Microneedles, pulmonary administra1on
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Subunit vaccines
Protein subunits
Diphteria, tetanus, hepa11s B
Bacterial polysaccharides (outer membrane)
Streptococcus pneumoniae, Nisseria meningi7dis, Haemophilus
inuenzae, Salmonella typhi
Viral glycoproteins
Herpes simplex virus
Synthe1c pep1des
From ac1ve epitopes of an1gen
HIV, inuenza, hepa11s B
Currently under inves1ga1on; not as immunogenic as proteins
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Memory of immune system

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Stabilisa9on of proteins (vaccines)

Vaccines (more general: proteins) in aqueous solu1on are
osen unstable
Oxida1on, hydrolysis, deamina1on, aggrega1on, denatura1on

Dry for longer shelf-life, without damaging the protein
Use excipients that stabilise proteins during drying process and in dry
state (storage)

Sugar glasses (amorphous sugar matrices) in which vaccine

(protein) is enclosed
Protec1on
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Which type of sugar for sugar glasses?

High glass-rubber transi1on temperature (Tg)
To avoid crystallisa1on
Slow crystallisa1on
Temperature, moisture, process stress
Low hygroscopicity
Water lowers Tg (acts as plas1ciser)
No or limited number of reducing groups (Maillard reac1on)
Flexible molecular structure
Non-toxic
Accepted by health authori1es
Cheap and readily available
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Mechanism of stabilisa9on
Vitrica1on theory (shielding)
Bulk eect
Water replacement theory
Sugar must be in amorphous state (1ght) and glassy (no diusion)
Sugar acts as barrier between protein par1cles
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Maillard reac9on
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Flexible backbone
Most oligosaccharides: rigid backbone
Dextran, maltodextrin
Inulin: exible backbone
CH2OH H
O
CH2OH
H OH
H O H
H CH2OH
OH H O
OH O
OH H
H OH CH2 CH2 H
H H O
O
H
OH H H OH
OH O CH2OH
n O
H OH CH2
CH2OH n
H O H OH H
H H CH2
OH H O H H
OH O
OH H
OH H
H OH H OH
OH O CH2OH

H OH
OH H
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Flexible backbone hypothesis

The consequence of the exible backbone for protein
stabilisa1on

Naked drug

Protec1on by Protec1on by
rigid oligomer exible oligomer
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Produc9on process
water + sugar water + protein

mixture
Freeze drying
Spray drying
Spray-freeze drying

N2(l)
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States of maXer
Thermodynamically stable Thermodynamically unstable
Crystal Glass
Molecules arranged in a lazce Random orienta1on of the molecules
Low transla1onal mobility of the (amorphous)
molecules Low transla1onal mobility of the molecules
Kine1cally stable: crystallisa1on can not occur
Liquid
Random orienta1on of the Rubber
molecules Random orienta1on of the molecules
High transla1onal mobility of the (amorphous)
molecules High transla1onal mobility of the molecules
Kine1cally unstable: crystallisa1on can occur
Super-cooled liquid
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Phases and states

Liquid Rubber

Crystal Liquid
T > Tm Crystal Glass
Glass Rubber
T > Tg
Tg << Tm
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Phase diagram sugar and water Thermo-

dynamically
Tm,sugar
stable
Temperature Homogeneous
Crystallisa9on solu9on Crystallisa9on
curve of water curve of sugar

Tm,water (273 K)
Te Two phases:
sugar crystals and
aqueous sugar
Two phases: solu9on
Two phases: ice + sugar crystals
ice and aqueous
sugar solu9on Pure water Pure sugar

Only when in complete equilibrium

In other cases: state diagram
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State diagram sugar and water Thermo-

Crystallisa9on dynamically
Temperature curve of sugar Tm,sugar unstable

Homogeneous solu9on
Crystallisa9on Rubber
curve of water
Tg, pure sugar

Tm,water (273 K)

Tg
Glass transi9on
curve

137 K Ice + Glass (Cg)

Pure water Cg Pure sugar

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Spray dried par9cles

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Inulin glass stabilisa9on

Technique applied to
Proteins Complex/par1cular
Alkaline phosphatase structures
Inuenza subunit vaccine Liposomes
Insulin Whole inac1vated
inuenza virus
rhDNAse
Virosomes
Iniximab
Cyclosporin A
Oxytocin
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Inuenza vaccine
Causes u
Enveloped virus
Hemagglu1nine (HA) mediates binding and fusion
Subunit vaccine: HA and Neuraminidase (NA)
HA is highly instable
Injec1on: shelf-life 6 months at 4C
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Whole inac9vated inuenza virus

Dry powder sugar glass

Eur J Pharm Biopharm 2013;85:716-25 remains stable
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Inuenza vaccina9on via inhala9on?

Viruses mutate: annual revaccina1on necessary
Pandemic viruses (avian u, Mexican u)
Vaccine: inac1vated or pure HA and NA of three dierent
virus types
Problems: poor stability of aqueous injec1on, fear of
injec1ons, injected vaccine only ac1ve in 50-70% of people
>65 years
Inhala1on of vaccine would mimic natural route of contagion

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Pulmonary powder administra9on (mice)

PennCentury insuator
Powder is ac1vely administered using 200 L air pus
Large amounts possible (up to 2 mg)
Par1cle size of aerosol comparable to Twincer DPI
Groningen inhala1on box
Aerosol generated with Twincer
Mul1ple administra1on
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Immune response (mice)

6
10log serum-IgG titer

Nose IgG * p<0.001

Lung

5 3

2 *p<0.05
4 6/8

log Ig titer
3 1/8 5/8

0
2 IgA

10
3 * p<0.001

intra i.m. a.i.

aerosol p.i. powder
muscular inhalation inhalation 2
* p<0.01
7/8
12 1
* p<0.01 2/8
10 0
i.m. a.i p.i.
log HI titer

8
Adjuvant ac1on of inulin
6

4
Protec1on at port of entrance:
2

2
also response in nose and long
0
i.m a.i. p.i. mucosa
Vaccine 2007;25:8707-8717
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An9bodies (1)
Proteins produced in B cells (lymphocytes)
Part of (acquired) immune system
- IgG, IgA, IgM, IgE, IgD
- To iden1fy specic foreign species
Medically applied
To obtain passive immunity
To neutralise pathogens in blood pa1ent
Conven1onally produced from blood of immunised animals
or humans
An1serum with high 1ter an1bodies
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An9bodies (2)
Polyclonal an1bodies
Mixture of an1body cells from all plasma cell clones that reacted to a
par1cular an1gen
Made from several dierent immune cells
Monoclonal an1bodies
Produced in iden1cal immune cells (ar1cially)
Single species of desired an1body
Same target object, binding to the same epitope
Wide range of medical applica1ons
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Monoclonal an9bodies
First genera1on
Murine
Immune response in 50-70% recipients
Short half-life
Inability to ac1vate human complement

Second genera1on
Engineered: chimeric, humanised, human
Half-life enhanced (up to 5-fold)
Improved func1onality
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Structure an9body (1)

Y-shaped
Consists of two main domains
Fc constant domain
Fab an1body-binding domain
At 1p Fab region hypervariable regions that bind to an1gen

Chimeric
Murine Fc region replaced by human Fc
Humanized
Only murine hypervariable regions amained, remainder is human
Human
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Structure an9body (2)

Fac

Fc
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Produc9on of mabs
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2nd genera9on mabs

An9body Type Target Use
Iniximab Chimeric mab Tumour necrosis factor Crohns disease,
rheumatoid disease
Adalimumab Humanised mab Tumour necrosis factor Rheumatoid disease

Trastuzumab Humanised mab HER2 epidermal growth Breast cancer
facmor receptor
Palivizumab Humanised mab Respiratory syncy1al Respiratory infec1ons
virus in young children
Omalizumab Humanised mab Immunoglobulin E Immunoglobulin E-
mediated asthma

Source: Rang & Dales Pharmacology

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Nomenclature of mabs (1)

All monoclonal an1body names end with mab

Source
-o- : mouse
-u-: human
-xi-: chimeric
-zu-: humanised
-xizu-: chimeric humanised
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Nomenclature of mabs (2)

Substem preceding source of ab refers to target
Tumours, organ systems, infec1ous agents (bacteria, viruses)
-ci(r)-: circulatory system
-ne(r)-: nervous system
-li(m)- : immune system
-co(l)-: colonic tumour
-go(v)-: ovarian tumour
-ma(r)-: mammary tumour
-me(l)-: melanoma
-tu(m)-: miscellaneous tumour
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Tumour immunology
Tumour cells present an1gens not found in normal cells
Cancer specic an1gens
Target for an1bodies
Used for drug targe1ng
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Macrophages aXack tumour cell

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Nucleic acid drugs

An1sense nucleo1des
Small interfering ribonucleic acids (siRNA)
Genes
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An9sense
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siRNA: small interfering RNA

siRNA is short (20-24 bp) double stranded RNA

which inhibits transla1on by combining
with RNA-induced silencing complex
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Delivery of nucleic acid drugs

Poor plasma stability of DNA, siRNA, oligonucleo1des
Inability of larger polar molecule to cross plasma cell
membranes
DNA nega1vely charged at physiological pH
Entry to cell nucleus limited to cell division events

Vector required
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ATMPs
Advanced Therapy Medicinal Products
Innova1ve products combining aspects of medicine, cell
biology, science and engineering for the purpose of
regenera1ng, repairing or replacing damaged 1ssues or cells

Contain (or consist of) cells or 1ssues that have been subject
to substan1al manipula1on or that are not intended to be
used for the same essen1al func1on(s) in the recipient as in
the donor and that have proper1es for trea1ng or preven1ng
disease in pa1ents
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Categories ATMPs
Gene therapy medicinal products (GTMP)
Soma1c cell therapy medicinal products (SCTMP)
Tissue engineered products (TEP)

Regula1on EC 1394/2007 (The ATMP regula1on)

Custom made
For individual pa1ent, hospital sezng
Industrially made
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Gene therapy (1)

Products obtained through set of manufacturing processes
Aimed at transfer (in vivo or ex vivo) of prophylac1c,
diagnos1c or therapeu1c gene (piece of nucleic acid) to
human cells followed by in vivo expression

Vectors: viral, non-viral

The vector can also be included in human or animal cells
 | 69

Gene therapy (2)

In vivo strategy
Vector with therapeu1c gene injected (iv, into target 1ssue)
Ex vivo strategy
Cells removed from pa1ent
- Stem cells from bone marrow or circula1ng blood
- Myoblasts from biopsy of striated muscle
Treatment with vector with therapeu1c gene
Injected back into pa1ent
 | 70

Ideal vector
Safe
Ecient (high transfec1on eciency)
Insert therapeu1c gene into high propor1on of target cells
Selec1ve
Expression of therapeu1c protein in target cells but not viral proteins
Produces persistent expression
No need for repeated treatment
 | 71

Vectors for gene therapy

Viral (clinically applied)
Retroviruses
- Infect many dierent types of dividing cells, randomly incorporated
Adenoviruses
- Strong host response, inamma1on, short-lived expression

Non-viral (thus far only experimental)

Lipoplexes (posi1vely charged liposomes)
Microspheres (biodegradable)
Plasmid DNA (naked DNA)
- Can be used as vaccine
An1sense oligonucleo1des
siRNA
 | 72

Adenovirus vector
 | 73

Safety of viral vectors

May acquire virulence during use
Contain viral proteins
Immunogenicity
Can elicit inammatory response
Could damage host genome and interfere with cell cycle
Provoca1on of malignancy

S1ll limited clinical experience

No big problems (yet)
 | 74

Therapeu9c applica9ons
Especially in oncology
Gendicine (Glybera): recombinant adenovirus, restores wild type p53
Promising approaches
Restoring protec1ve proteins (p53)
Inac1va1ng oncogenes
Delivering a gene that renders malignant cell sensi1ve to drugs
Delivering gene to healthy host cell for protec1on against
chemotherapy
Tagging cancer cells with genes that make them immunogenic

Infec1ous diseases (vaccines), cardiovascular disease

 | 75

Hurdles in gene therapy

Delivery problems
Pharmacokine1cs: delivery of gene to appropriate target cells
Pharmacodynamics: controlled expression of gene in ques1on
Safety
Clinical ecacy
Long-term prac1cability
 | 76

Soma9c cell therapy

Autologous (coming from pa1ent), allogeneic (from other
human being) or xenogeneic (from animal origen) soma1c
living cells
Biological characteris1cs substan1ally altered by
manipula1on
To obtain therapeu1c, diagnos1c or preven1ve eect
Through metabolic, pharmacological and immunological means
Including treatment of autologous cell popula1ons ex vivo

Bone marrow transplanta1on

 | 77

Tissue engineered products

Contain or consist of engineered cells or 1ssues
Given to human beings to regenerate, repair or replace a
human 1ssue
May contain human and/or animal cells or 1ssues
Cells may be viable or non-viable
May contain cellular products, biomolecules, biomaterials,
chemical substances, scaolds, matrices
Engineered:
Not intended for use for same func1on(s) in recipient as in donor
Subject to substan1al manipula1on to achieve certain proper1es
 | 78

Examples of ATMP (EMA)

GTMP
Gene1cally modied adenovirus coding for human granulocyte-
macrophage colony s1mula1ng factor (GM-CSF)
- Treatment of cancer

SCTMP
Allogeneic mesenchymal precursor cells
- Rheumatoid arthri1s
Human autologous tumour-inltra1ng lymphocytes
- Stage III melanoma with one invaded lymph node

TEP
Human dermal broblasts cultured on bioresorbable polyglac1n mesh
- Treatment of wounds and ulcers
| 79