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1
HYDROXYPROPYL-ß-CYCLODEXTRINS
KLEPTOSE® HPB & HP:
NOVEL TOOLS FOR PROTEIN STABILIZATION
DDF San FranciscoSept 10-11th, 2018Sofiya Yashchuk
Global Technical Application Specialist - Biopharma
3
SUPPLIER OF PLANT DERIVED EXCIPIENTS & FUNCTIONAL INGREDIENTS FOR A HEALTHIER LIFESTYLE
3
Pharma Cosmetics IndustriesAnimal NutritionFood & Nutrition
• Dextrose
• Polyols
• Native & modified
starches
• Maltodextrins
• Glucose syrups
• Proteins
• Fibers
700+ solutions
77
https://www.pvd.co.nz/vet-pages/beta-cyclodextrin
7 (β-cyclodextrin) 6 (α-cyclodextrin) 8 (γ-cyclodextrin)
http://www1.lsbu.ac.uk/water/cyclodextrin.html
NATIVE CYLODEXTRINS
# of Glucopyranose units (n):
Cyclodextrins (CDs) are cyclic oligosaccharides obtained from
starch by enzymatic cyclisation using cycloglycosyl
transferases (CGTs)
88
https://www.pvd.co.nz/vet-pages/beta-cyclodextrin
7 (β-cyclodextrin) 6 (α-cyclodextrin) 8 (γ-cyclodextrin)
Cavity Volume M.W.Aq Solubility at 25 ºC
174 Å3, 0.10 mL/g972
129.5 g/kg H2O
262 Å3, 0.14 mL/g1134
18.4 g/kg H2O
427 Å3, 0.20 mL/g1296
249.2 g/kg H2O
http://www1.lsbu.ac.uk/water/cyclodextrin.html
NATIVE CYLODEXTRINS
# of Glucopyranose units (n):
9
NATIVE CYLODEXTRINS
• ß-CD is the most widely used CDs• The cavity size of α-CD is too small to accommodate most APIs
• γ-CD is too large & expensive to produce
• Due to low aq solubility native ß-CD is not suitable for injectable formulations• Substituted derivatives must be used instead
1010
Hydroxypropylated CDs
HP-β-CD
MS
sign
alM
S si
gnal
Batch-to-batch consistency at Roquette is rooted in a long manufacturing history of HPBCD –providing consistent pattern of substitution
M.W.
MS, DS
Solubility in H2O
Residual BCD
Kleptose® HPB~1390
0.65, 4.3>50% (20 ºC, w/w%)
0.7-0.8%
Kleptose® HP~1500
0.88, 6.7>50% (20 ºC, w/w%)
~0.1%
ROQUETTE MODIFIED CYLODEXTRINS
11
CYCLODEXTRINS MECHANISM EXPLAINED
• The fundamental basis of CDs in pharmaceutical applications is the capability to form
inclusion complexes due to the hydrophobic property of the cavity
• The drug-CD complexes hides most of the hydrophobic functionality of the drug active in
the interior cavity of the CD while the hydrophilic hydroxyl groups on its external surface
remained exposed to the environment
• The net effect is a water-soluble CD exposed to the environment
In small molecules pharmaceutical applications
Hydrophobic drug
Hydrophobic Cavity
Hydrophilic Exterior
• Secondary OH groups (C2, C3) on the wider edge &
Primary OH groups (C6)on the narrow edge
► Polar hydrophilic exterior
• Cavity lined by H and C atoms and etheroxyde bonds (glucosidic O bridges)
► Apolar hydrophobic cavity
12
HPßCDs are a powerful tool in small molecule formulation development
• Known in small molecules for years
• Improving solubility, chemical and physical stability, taste masking etc.
• Approved in small molecule injectable formulations
12
Hydroxypropyl-ß-Cyclodextrins – Small Molecule Applications
HP-β-CD / Ranitidine (Zantac) Complex
https://pubs.rsc.org/en/content/articlehtml/2018/ra/c7ra11015d
HP-β-CD / Darunavir Complex
13
MARKETED PHARMACEUTICAL PRODUCTS CONTAINING CDSSMALL MOLECULE
Drug/ Cyclodextrin Trade Name Dosage Form Company (country)
ß-CD
Piroxicam Brexin, Flogene, Cicladon Tablet, suppository Chiesi (Europe), Ache (Brazil)
Cetirzine Cetrizin Chewable tablet Losan Pharma (Germany)
Diphenhydramine and chlortheophylline
Stada-Travel Chewable tablet Stada (Europe)
Tiaprofenic acid Surgamyl Tablet Roussel-Maestrelli (Europe)
HPßCD
Indomethacin Indocid Eye drop solution Chauvin (Europe)
Itraconazole Sporanox Oral and intravenous solution
Jassen (Europe, USA)
Mitomycin MitoExtra, Mitozytrex intravenous solution Novartis (Europe)
SBEßCD
Voriconazole Vfend Intravenous solution Pfizer (USA, Europe, Japan)
Aripiprazole Abilify Intramuscular solution Bristol-Meyers Squibb (USA)
14
HPßCD IN PARENTERAL APPLICATIONS
• HPßCD is one of the two ß-CD derivatives (the other being 2-sulfobutylether-ß-CD) that are approved for used in parenteral products so far.
• HPßCD is used in approved parenteral products such as Itraconazol(Sporanox®) and Mitomycin (MitoExtra®).
• Extensive toxicological and pharmacological studies of HPßCD show that it issafe for parenteral application.
• An intravenous (IV) injection of HPßCD given as a single dose as high as 3.0 gis found to be safe and well tolerated in a human clinical study.
• Currently marketed parenteral products contain exclusively small moleculeAPIs.
• However, due to its status as an approved parenteral excipient, HPßCD hasgained great attention as potential excipient for protein formulations.
15
• Protein aggregation is a major challenge encountered during manufacturing,storage, processing, transportation and administration of biologics to patients.
• The presence of aggregates can severely influence the pharmacokinetics aswell as the safety of the protein drug.
• In the case of protein drugs, the mechanism of CDs is somewhat different fromsmall molecule drugs. Complexation does not involve encapsulation of theentire macromolecule.
HPßCD AS EXCIPIENT FOR PROTEIN FORMULATIONS
Partially Unfolded Protein Aggregated Proteins
UnfoldingAggregation
Refolding
16
Serno et al. Protein stabilization by cyclodextrins in the liquid and dried state. Advanced Drug Delivery Reviews 63 (2011) 1086–1106.
HPßCD – Proposed Protein Stabilization Mechanisms
• Binding to exposed hydrophobic resides on proteins,
mitigating hydrophobic interactions and thereby blocking
potential protein-protein interaction which lead to
aggregation.
• The cavity diameter of ß-CDs derivatives allows for a
fit of Phe, Tyr, His and Trp into the hydrophobic moiety.
• Inhibiting protein aggregation induced by exposure to air-
water interface.
• HPßCD could inhibit protein aggregation in a similar
manner to non-ionic surfactants, by displacing proteins
from the air-water interface.
• HPßCD shows surface activity at concentration of
0.1% (w/v).
protection by complexation with exposed hydrophobic residues
HPßCD can act as inhibitor of protein aggregation in liquid formulations by:
17
REPORTED APPLICATIONS OF HPβCDPromising results reported in various scientific publications
1. Reduce interfacial induced aggregation
Model protein studied Author (Year)
Rh-GH Tavornvipas et al. (2004)
IgG mAb from Roche Serno et al. (2010)
2. Reduce thermal denaturation
Model protein studied Author (Year)
Mink growth hormone Bajorunaite et al. (2009)
Creatine kinase Maloletkina et al. (2010)
Salmon Calcitonin Sigurjondottir et al. (1999)3. Reduce chemical denaturation
Model protein studied Author (Year)
Rh-GH Otzen et al. (2002)
Recombinant mink and
porcine growth hormoneBajorunaite et al. (2009)
Aminoacyclase Kim et al. (2006)
4. Lyoprotectant
Model protein studied Author (Year)
LDH Iwai et al. (2007)
Mouse IgG2a
mAbRessing et al. (1992)
5. Protein solubilizing effect
Model protein studied Author (Year)
Ovine growth hormone Brewster et al. (1991)
Interleukin-2 Brewster et al. (1991)
18
REPORTED BIOPHARMA APPLICATIONS OF HPΒCD
18
Model Protein CDs Other excipients Stress Methods Key Results Author (year)
Reduce interfacial interaction
IgG mAb
from Roche
CDs
HPbCD, MßCD, HPgCD and
SBEßCD
Sugars, polyols and surfactant
Tween 80, trehalose, sucrose,
mannitol and sorbitol
Agitation
Superiority of HPβCD to trehalose, sucrose,
mannitol and sorbitol or polysorbate 80 in low
concentrations.
Serno et al.,
2010
Lyoprotectant
LDH (12.8 μg/ml)
CDs
α-CD, HPαCD, β-CD, HPβCD,
SBEβCD, MβCD, γ-CD, HPγCD
Sugars
Glucose, maltose, trehalose,
sucrose, raffinose
Lyophilized and reconstituted
with purified water
HPβCD is more effective protein stabilizers than
low molecular weight saccharides. HPβCD
superior to trehalose.
Iwai et al.,
2007
Mouse IgG2a
mAb (1.0 mg/ml)
CDs
HPβCD (5% w/v)
Sugars
Sucrose, dextran
Lyophilized, reconstituted and
storage at 4, 37 and 56 °C
HPβCD comparable to sucrose and dextran after
lyophilization but superior during storage at 56 °C
Ressing et al., 1992
IgG monoclonal antibody
(20 mg/mL)HPβCD (80 mg/mL)
Sugars
TrehaloseSupercritical fluid drying HPßCD us as efficient as trehalose Jalalipour et al., 2008
ß-Galactosidase
(4% w/v)
CDs
HPβCD (1% w/v)
HPßCD (+ sucrose)
Sugar
SucroseSpray-drying HPßCD superior to sucrose Branchu et al., 1999
CYP3A4
CDs
MßCD, HPßCD (0.003 -0.034 %
w/v)
Sugars
2 crown ethers, sucrose,
trehalose, mannitol
LyophilizedCDs inferior to sugars, slight effect
at very low concentrationsChefson et al., 2007
A comparison of HPßCD with other sugars and polyols
2020
• Evaluation of aggregation reduction by HPßCDs• Kleptose HPB® (MS 0.62, DS 4.3) & Kleptose HP® (MS 0.9, DS 6.7)
• 2 Model Proteins Prone to Aggregation:• Human Growth Hormone (hGH), as a model
• Infliximab, a chimeric IgG1 (mAb), sold as a lyophilized powder
https://www.researchgate.net/figure/Infliximab-Remicade-protein-structure_fig1_303483394
Pavlovsky et al. The crystal-structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept Lett. 1995;2:333–340.
hGH Human Growth Hormone22 kDa
Infliximab149 kDa
STUDY DESIGN
21
• iFormulate® : A High Throughput DOE Approach to Formulation Development
• Pre-made formulations in statistically relevant and random design
• High Throughput Label-free nanoDSF screening
• Based on tryptophan fluorescence of exposed hydrophobic residues
21
EXPERIMENT TECHNIQUESTUDY DONE AT HTD BIOSYSTEMS INC
Pre-designed formulation plates
(20 formulations + 5 replicates) for the evaluation of
4 variables in multivariate DOE design:
1. pH
2. Ionic Strength
3. Buffer Concentration
4. Excipient ConcentrationiFormulate® Plate
1. Add protein to wells (< 10 mg required per 25 wells)
2. Stress protein formulations (e.g. thermal, F/T, etc.)
3. Sample Analysis by nanoDSF (Tm, Aggregation Onset Temp)
4. Data Analysis using DOE software
5. Optimize and Rationalize the formulation
22
Multiple stability and conformational parameters from one sample
22
DSF and Aggregation profiles of mAb in different formulations
DSF profiles
Tm’s
Onset T of Aggreg.
Output Data :
NanoDSF : indicates protein unfolding
Melting temperature (Tm)
Usually the higher, the more stable
Light scattering : indicates protein
aggregation
Relative amount of aggregation
(Agg_temp)
The higher, the more aggregated
Onset of aggregation (Agg_Onset)
Usually the higher, the more stable
DATA OUPUT
2323
EXPERIMENT METHOD
4. Ranking of key
variables of formulation
1. Plate prep 2. nDSF analysis
with thermal ramp3. Data collection
5. Predictive
mathematical model
(response-surface)
6. Defining optimal
formulation space
OBJECTIVES
VARIABLES
& MODEL
CHECK
POINTS
REPORT
RESOLVING
POWER
DATA
COLLECTIONANALYSIS
DESIGNGRAPHS
& TABLES
Experiments By Design Process
24
• DOE variables
opH 4 – 7.6
oNaCl 0 – 200 mM
oBuffer 10 – 50 mM
oStabilizing Excipient HP/HPB 25 – 200 mM
• Test Thermal Stability of hGH and Infliximab proteins under thermal ramp to 90ºC in the presence of stabilizer
o20OC to 90OC at 1.5 degrees/min
24
STUDY PARAMETERS
2525
Conditions :
• hGH = 4 mg/mL in
water
• 100 mM NaCl
• Fixed pH
• Increasing HP
concentration
• Suppression of aggregation observed • Reduction of aggregation ( mAU), higher melting temperature ( Tm)
• Similar results with both Kleptose HP ® & HPB ®
KLEPTOSE HP® IMPACT ON hGH
26
• DOE Results and Analysis
26
• Ionic strength and pH are key variables for hGH aggregation
• At high pH (7-7.6) HP reduces protein aggregation
• Similar trends for HP and HPB
Tm Tm Tm
Tm
Tm Tm Tm
Agg
rega
tio
n
Pareto effect for Agg. Onset temperature and 3D-plots showing the effect of NaCl and HP, pH and HP, and NaCl and pH on T Agg Onset temperature
Pareto effect for Tm and 3D-plots showing the effect of NaCl and HP, pH and HP, and NaCl and pH on Tm
KLEPTOSE HP® IMPACT ON hGHDOE Results and Analysis
2727
Increasing Temperature
• Suppression of aggregation observed • Reduction of aggregation ( mAU)
• No contribution to the thermal stability for Tm detected under these conditions
• Similar results with both Kleptose HP ® & HPB ®
KLEPTOSE HPB® IMPACT ON INFLIXIMAB
Control
4 mg/mL Infliximab in water
28
KLEPTOSE HPB® IMPACT ON INFLIXIMAB
28
DOE Results and Analysis
• pH observed to be the most important variable for Infliximab aggregation
• HPBCD impacted protein aggregation at low and high pH (quadratic effect)
• Similar trends observed for HPB and HP
TmA
ggre
gati
on
Tm Tm Tm
Tm Tm Tm
29
• Under Stressed Conditions
29
100
120
140
160
180
200
220
240
260
pH7.6NaPO4 HP-100 HPB-100 wHP-100 wHP5-100 wHP7-100
hGH Agg at 85 ºC
100
120
140
160
180
200
220
240
260
280
pH7.6NaPO4 HP-100 HPB-100 wHP-100 wHP5-100 wHP7-100
Infliximab Agg at 90 ºC
Conditions : 4 mg/mL protein in pH 7.6 NaHPO4 buffer, 100 mM for each Kleptose
Relative Degree of Aggregation at 100 mM HP® & HPB®
Back S
cattering
(m
AU
)
• Kleptose HP ® & HPB® Reduce Aggregation
• HPB ® offers stronger performance under these conditions
100 mM NaCl
30 mM buffer 200 mM NaCl
10 mM buffer
KLEPTOSE HP ® & HPB® COMPARISON
30
• Kleptose HPB® & HP® showed stabilization affects on proteinsHuman Growth Hormone & Infliximab: reduced relative amount of aggregation
30
• Kleptose HPB® & HP® show similar trends with the 2 studied proteins
Selection between HPB and HP grades may be protein dependent. Sampling of both grades is advised
• Further studies ongoing at the Roquette Innovation Center, Singapore
CONCLUSIONS
31
• Sofiya Yashchuk – Global Technical Application Specialist
• Mark Driesner – Biopharma Business Developer – North America
• Eva Esparza – Biopharma Business Developer – Europe
31
For product sampling and technical support,
please contact our dedicated Biopharma team:
GLOBAL CONTACTS
34
REPORTED BIOPHARMA STUDIES WITH HPßCD
34
Effect of Cyclodextrins on protein aggregationAdvanced Drug Delivery Reviews 63 (2011) 1086–1106;Protein stabilization by cyclodextrins in the liquid and dried state ;Tim Serno, Raimund Geidobler ,Gerhard Winter
35
REPORTED BIOPHARMA STUDIES WITH HPßCD
35
Effect of Cyclodextrins on protein aggregation continued…
Advanced Drug Delivery Reviews 63 (2011) 1086–1106;Protein stabilization by cyclodextrins in the liquid and dried state ;Tim Serno, Raimund Geidobler ,Gerhard Winter
36
HPßCD – STABILIZING MECHANISMS AS LYOPROTECTANT
Reported benefits of HPßCD as excipient for freeze- and spray- drying of protein solutions:
Protein stabilization by non-ionic surfactant-like behaviorAt low CD concentrations
(i.e. 0.0001 – 1 % (w/v))
• Inhibiting protein unfolding and aggregation at the interfaces (i.e. air-water, ice-water)
Protein stabilization by water replacement and vitrification
At high CD concentrations(i.e. Weight ratio of at least 1:1)
• Serves as water substitute (i.e. lyoprotectant) via hydrogen bonding
• Yields amorphous glass during freeze- or spray-drying. Protein is immobilized in the matrix and kinetically stabilized
High glass transition temperatureDuring storage of lyophilizates
• Increase storage stability of CD-containing lyophilizates can be attributed to the high glass transition temperatures, Tg of CDs
3737
Mission
To serve patients globally by providing
safe ingredients and raw materials for the
Pharmaceutical and Biopharmaceutical
industries.
ROQUETTE PHARMA
Our quality standards are
uncompromised and our products
have a transparent and secure
supply chain from raw material to the
finished product delivered to the
clients.
3838
> 30 years in
pharma market
#1supplier of active
ingredient (dextrose)
for dialysis and I.V.
products globally
#1supplier of
mannitol and sorbitol
as excipients in oral
dosage forms and
parenteral products
9 manufacturing
locations
R&D / Customer Technical Support
• In-house laboratories
– U.S., France,
Singapore
• External –customer sites
Certifications
• EXCiPACT
• ISO 9001
• CGMP
Quality Management Systems
• ISO 9001
• 21 CFR part 210/211
• European GMP, Eudralex
Volume 4, part II
• ICH Q7
• EXCiPACT
GovernmentRegulatoryRegistrations
• FDA #1000114558
• ANSM (French
Health Authority)
ROQUETTE PHARMA
3939
FAMILY FOUNDED & OWNED COMPANY
Owned by
over 200 family shareholders
from the 2nd to the 5th generation
Founded by
Dominique & Germain Roquette
1933
Today
4040
5,000+Customers
1Global commercial
network in more
than 100 countries.
85 Years of industrial
and operational
excellence.
8,400Employees
45+Nationalities
20 Industrial sites
40/yearsPatents
€3.3bnTurnover
300+ R&D workforce
Roquette frères 2018
4141
A GLOBAL PRESENCE TO SERVE OUR CUSTOMERS
• A global offer • Competitive
everywhere
• Locally
customised
• With uniform
standards
PANTNAGAR LIANYUNGANGKEOKUK LESTREM
4242
DELIVERING OUR EXPERTISE TO TAILOR SOLUTIONS FOR CUSTOMERS
Enzymatic
engineering
Fermentation
Mannitol
Soluble fiber
Sorbitol
Modified starch
Microalgae
Continuous
chromatography
Maltitol
Cyclodextrin
Isosorbide
Pea protein
Low calorie
sugar
Innovating Since 1933
Xylitol
• A network of innovation centers to partner with customers and get
products to market
• Above industry standards
> A strong platform of innovative products and processes
4343
STARCHStarch molecule
= Thousands of glucose
molecules linked together
DryingNative starch
Modification
Modified starchDextrins, Pregels,
Ethers-esters, Cationics,
other
Hydrolysis
Enzymes
100
20 40 80
Products from hydrolysis
Maltodextrins Glucose syrups Hydrolysates
Dextrose
Fermentation Hydrogenation
Products from fermentation
Products from hydrogenation
Organic acids Hydrogenated
glucose syrups
Polyols: sorbitol,
mannitol, maltitol, xylitol
Isosorbide
sugars
Isosorbide
derivatives
where we are currently active
STARCH AND DERIVATIVES
4444
Process Dextrose
Anhydrous
Dextrose
Monohydrate Sorbitol Mannitol
HPBCD(hydroxyl propyl
modified
cyclodextrins)
Cell Culture
Solutions
Protein
Stabilisation
Solutions
•Additional testing beyond the monograph includes: Beta Glucans, DNAse,
Protease, Residual DNA, ICH Q3D, and Pesticides
•Rx360 Supplier Assessment Questionnaire and Technical Information
Packages are available
•All products are of non-animal origin (plant derived)
ROQUETTE BIOPHARMA SOLUTIONS
4545
Endotoxin level ≤ 5 IU/g
No-observed adverse effect
level (NOAEL) = 320 mg/kg
body weight per day
Endotoxin level ≤ 1 IU/g
http://www.ema.europa.eu/docs/en_GB/document_library/Report/2014/12/WC500177936.pdf
PROTEIN STABILIZATION SOLUTIONS