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HYDROXYPROPYL-ß-CYCLODEXTRINS

KLEPTOSE® HPB & HP:

NOVEL TOOLS FOR PROTEIN STABILIZATION

DDF San FranciscoSept 10-11th, 2018Sofiya Yashchuk

Global Technical Application Specialist - Biopharma

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INTRODUCTION TO ROQUETTE

2

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

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DEDICATED PHARMA SOLUTIONS

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Protein Stabilisation

ROQUETTE BIOPHARMA SOLUTIONS

Cell Culture

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OVERVIEW OF

ß-CYCLODEXTRINS

6

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)

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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

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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

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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

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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

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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)

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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.

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• 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

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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:

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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)

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REPORTED BIOPHARMA APPLICATIONS OF HPΒCD

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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

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CASE STUDY:

PROTEIN STABILIZATION BY KLEPTOSE HP & HPB

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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

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• 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

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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

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Multiple stability and conformational parameters from one sample

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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

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• 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

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STUDY PARAMETERS

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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

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• DOE Results and Analysis

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• 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

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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

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KLEPTOSE HPB® IMPACT ON INFLIXIMAB

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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

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• Under Stressed Conditions

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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

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• Kleptose HPB® & HP® showed stabilization affects on proteinsHuman Growth Hormone & Infliximab: reduced relative amount of aggregation

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• 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

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• Sofiya Yashchuk – Global Technical Application Specialist

• Sofiya.Yashchuk@roquette.com

• Mark Driesner – Biopharma Business Developer – North America

• Mark.Driesner@roquette.com

• Eva Esparza – Biopharma Business Developer – Europe

• Eva.Esparza-Guijarro@roquette.com

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For product sampling and technical support,

please contact our dedicated Biopharma team:

GLOBAL CONTACTS

32

THANK YOU

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APPENDIX

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REPORTED BIOPHARMA STUDIES WITH HPßCD

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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

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REPORTED BIOPHARMA STUDIES WITH HPßCD

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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

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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

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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

4646

Lycadex® BioPharma Endotoxin ≤1 IU/g

- Suitable for use in cell culture and injectable applications

- JP monograph now harmonized with EP

Dextrose Anhydrous BioPharma

- Suitable only for cell culture applications

CELL CULTURE SOLUTIONS