1

HYDROXYLPROPYL ß-CYCLODEXTRIN: A PROMISING EXCIPIENT FOR PROTEIN

STABILIZATION

DDF 2019, Berlin

Rajeev Gokhale

212 Mar 2019 Global Drug Delivery and Formulation Summit 2019

ABSTRACT

• Protein stability is a major challenge to overcome during manufacturing, upstream and downstream processes, formulation, transportation and storage of biopharmaceuticals. Proteins may aggregate during these activities in addition to chemical changes. Aggregation is often irreversible leading to inactive and potentially immunogenic species, resulting in to reduced efficacy and toxicity to patients. Formulation development involves selection of appropriate excipients to stabilize the protein drugs throughout its recommended shelf life against potential excursions in its life cycle and aid in the delivery of therapeutics into the patient. While role of many excipients in the stability of formulation is well documented, limited information is available on the role of Cyclodextrins in stabilization of therapeutic proteins such mAbs. Cyclodextrins play a vital role in reduction of interfacial tension with the environment and hydrophobic interactions among the protein molecules. In the present study stabilizing effects of novel Cyclodextrins, KLEPTOSE® HPB and HP in various therapeutics protein formulations have been investigated. Physical-chemical stability was influenced under different stress conditions such as thermal and agitation for monoclonal antibodies and human growth hormone. We employed a two-prong approach, high throughput screening and conventional chromatographic methods to establish validation. This study provides insight in to both methodologies to understand interplay between protein and surrounding environments. We attempt to provide the role of novel Cyclodextrins to reduce aggregation and develop therapeutically effective and safe protein formulations

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✓ Proteins are inherently unstableProteins are sensitive to temperature changes, shearing, shaking, solvents, ionic strength, purity, protein concentration, pressure, freeze/thaw-drying cycles. They are generally stable in narrow pH range, and are susceptible to adsorption.

✓ Of the various degradation pathways, aggregation is one of the most common and a cause for great concern.

Protein aggregates reduce drug efficacy and can induce immunogenicity (!)

✓ Formulation is a key element of a successful, stable protein drug. By identifying optimalsolution conditions such as pH, ionic strength, and excipients, the propensity for degradationcan be reduced (Goldberg et al., 2011).

CHALLENGES IN PROTEIN FORMULATIONS

44

PROTEINS UNDERGO CHANGES DURING PROCESSES, STORAGE AND TRANSPORTATION

SN Stress Factors

Process/storage/transportation conditions

1 Elevated Temp

Production, improper shipment, storage or handling or deviations, specific production processes like spray drying

2 Freeze-thaw

In process storage, accidental freezing during shipment, Lyophilization

3 Mechanical stress

Production process; shipping (pumping, filtering, stirring, shaking)

4 Light Exposure to day light during production processes, shipping, handling; UV detection during DSP unit operations

5 Oxidative stress

Contacts with oxygen (air, dissolved oxygen), excipients-e.g. peroxides in Polysorbates

6 pH change Production processes (different loading and elution conditions in chromatographic purifications), freezing, formulation

7 Interfaces Air water interface, filters, primary packaging, silicone oil

8 Radiations X-ray in air freight transportation

512 Mar 2019 Global Drug Delivery and Formulation Summit 2019

FORMULATION DESIGN

Buffer

Excipient 3

Tune viscosityExcipient 2

Enhance solubility

Excipient 1 Prevent aggregation

Surfactant Reduce interfacial tension

Ensure initial stability

Solubility

Long-term stability

(aggregation)

Viscositye.g. Polysorbates, poloxamer

e.g. hydroxypropyl cyclodextrin

e.g. arginine

e.g. sugars, polyols, hydroxypropyl cyclodextrin

e.g. citrate, histidine, phosphate

Formulation Challenges

6

CONCERNS IN THE USE OF POLYSORBATES

Autooxidation of ethylene oxide

Peroxides

Oxidation of protein

Protein aggregation

❑ Polysorbates are the most widely used non-ionic surfactants

• > 70 % the marketed formulations of monoclonal antibody products contain either polysorbate 20 or 80.

❑ Protect protein pharmaceuticals against interface-induced

aggregation and surface adsorption

❑ Polysorbates 20 and 80 are chemically diverse mixtures• contain mainly sorbitan polyoxyethylene esters of fatty acids• prone to degrade by autooxidation and hydrolysis• presence of residual levels of peroxide in bulk

❑ Polysorbates degradation concerns:

lowered ability of the surfactant to protect the formulation against interfacial stressesimpact of the degradation products on the stability of the protein.

Polyoxyethylenemoieties

77

HYDROXYPROPYL-ß-CYCLODEXTRINS

❑ CDs and their derivatives are established enabling excipients in small molecule formulation development

➢ Solubility and bioavailability enhancement

➢ Improve drug chemical and physical stability

➢ Taste-masking

Hydroxypropyl-ß-Cyclodextrin

Hydrophobic Cavity

Hydrophilic Exterior

Unique structure allows for the formation of inclusion

complexes, where lipophilic compounds are

non-covalently bound within the cavity

M.W.

MS, DS

Solubility in

H2O

KLEPTOSE® HPB~1390

0.65, 4.3>50% (20 ºC, w/w%)

KLEPTOSE® HP~1493

0.88, 6.7>50% (20 ºC, w/w%)

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HPßCD – PROTEIN STABILIZING MECHANISMS

❑ HPßCD can act as inhibitor of protein aggregation in liquid formulations by:

i. Binding to the exposed hydrophobic resides on the proteins. This binding lead to shielding of hydrophobic interactions and thereby blocking potential protein-protein interaction which cause aggregation.

➢ The cavity diameter of ß-CDs derivatives allows for a fit of Phe, Tyr, His and Trp into the hydrophobic moiety.

ii. 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).

12 Apr 2017 Confidential

Protein

Reference : Serno, T., R. Geidobler, and G. Winter, Protein stabilization by cyclodextrins in the liquid and dried state. Adv Drug Deliv Rev, 2011. 63(13): p. 1086-106.

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0

100

200

300

400

500

600

700

800

900

0 50 100 150 200 250

Osm

ola

lity

(mO

smo

l/kg

)

Concentration (mM)

Osmolality of KLEPTOSE®HP/HPB

KLEPTOSE®HP

KLEPTOSE®HPB

▪ Usage of KLEPTOSE HP/HPB at concentrations as high as 250 mM will not impart high viscosity to protein solutions

▪ Acceptable viscosity for SC injections is ~1-20 cps

▪ Recommended upper osmolality limit for SC injection: under 600 mOsm/kg

▪ Usage of KLEPTOSE HP/HPB at high concentrations (i.e. above ~100-200 mM) may be acceptable

PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPB

Osmolality and Viscosity

PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPB

0

20

40

60

80

100

0 100 200 300 400 500V

isco

sity

(m

Pas

)

Concentration (mM)

Viscosity of KLEPTOSE®HP/HPB

KLEPTOSE®HP

KLEPTOSE®HPB

M.W.

MS, DS

Solubility in H2O

KLEPTOSE® HPB (Biopharma grade)~1390

0.65, 4.3>50% (20 ºC, w/w%)

KLEPTOSE® HP (Biopharma grade)~1493

0.88, 6.7>50% (20 ºC, w/w%)

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PHYSICAL PROPERTIES OF KLEPTOSE®HP/HPBSurface Tension

50

55

60

65

70

75

0 50 100 150 200 250

Surf

ace

Ten

sio

n (

mN

/m)

Concentration (mM)

Surface tension of KLEPTOSE®HP/HPB

KLEPTOSE®HP

KLEPTOSE®HPB

MS~0.88

▪ KLEPTOSE®HP/HPB showed surface-active properties.▪ Higher HP substitution, lower surface tension. ▪ Critical concentration of KLEPTOSE®HP is ~ 3-4 mM and HPB is ~ 10 mM.▪ Critical micellar concentration (CMC) of polysorbate 80/20 is 0.012 mM and 0.08mM,

respectively

MS~0.65

1111

• High throughput: Multiple stability and conformational parameters from one sample

AGGREGATION STUDY OF KLEPTOSE® HP & HPB: NANODSF

Drawbacks: Study aggregation profiles of protein at thermal unfolding temperatures (i.eabove Tm). Protein aggregation propensity might be different in real-time stability studies.

Fluorescence detection at 350 nm and 330 nm→ measures protein unfolding profile

Light scattering → measures relative amount of aggregation

DSF and Aggregation profiles of mAb in different formulations

Unfolding profiles

Tm’sOnset T of Aggreg.

F 3

50

/33

0 r

atio

Bac

k Sc

atte

rin

g

Relative amount of aggregation

Temp. ramp

1212

• Thermal ramp analysis using nanoDSF

https://www.researchgate.net/figure/Infliximab-Remicade-protein-structure_fig1_303483394

Human Growth Hormone (hGH)22 kDa

Infliximab149 kDa

PREVIOUS STUDY : Evaluation of aggregation reduction by KLEPTOSE® HP & HPB

Ba

ck S

ca

tte

rin

g (

mA

U)

Ba

ck S

ca

tte

rin

g (

mA

U)

➢ Suppression of aggregation observed in the presence of KLEPTOSE® HP & HPB

https://www.researchgate.net/figure/Infliximab-Remicade-protein-structure_fig1_303483394Pavlovsky et al. The crystal-structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept Lett. 1995;2:333–340.

Increasin

g HP

B

hGH Agg at 85°CInfliximab Agg at 90°C

Temperature Temperature

Buffer HP 100 mM HPB 100 mM Buffer HP 100 mM HPB 100 mM

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https://www.drugbank.ca/drugs/DB00112

CASE STUDY 1: PROTEIN STABILIZATION BY KLEPTOSE ® HPB & HP

Objective: To benchmark KLEPTOSE ® HP/ HPB against polysorbates in commercial monoclonal

antibodies formulations.

Human Plasma IgG~150 KDa

Model proteins:

Commercial formulation:25 mg/mL bevazizumab, 60 mg/mL α,α-

trehalose dihydrate, 50 mM phosphate buffer, pH 6.2, 0.04 % w/v polysorbate 20

▪ Marketed as Avastin, is a recombinant

humanized monoclonal antibody (IgG1) used to treat a number of types of cancers

Bevacizumab149 KDa

https://www.drugbank.ca/drugs/DB00112

▪ Immunoglobulin G (IgG) is the most abundant

immunoglobulin in plasma

Proposed base buffer used in study:25 mM phosphate buffer, pH 6.8

https://www.differencebetween.com/difference-between-igm-and-vs-igg/

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EXPERIMENTAL PLAN

Controls

Buffer only

0.05% Tween 80

with [HPßCD]

Shaking @ 1400 rpm Incubation at 40 °C

1) NanoDSF:▪ Aggregation Onset

Temperature ▪ Relative amount of

aggregation

2) HPLC-SEC ▪ % soluble monomers,

aggregates & fragments

Human plasma IgG:

Bevacizumab (Avastin):Controls

CF

CF w/o Tween 20 + [HPßCD]

5 mM

20 mM

100 mM

Formulation Strategy: ➢ All formulations will be formulated at 5 mg/mL protein

concentration ➢ For Human plasma IgG, polysorbate 80 (Tween 80) at 0.05

%w/v level (above CMC) will be used as control➢ For bevacizumab, commercial formulation (CF) with/ without

Tween 20 will be used as controls. ➢ Effect of KLEPTOSE ® HP/ HPB was studied at 3 different levels

(i.e 5 mM, 20 mM and 100 mM)

CF

w/o Tween

20

5 mM

20 mM

100 mM

Two-pronged approach

1512 Mar 2019 Global Delivery and Formulation Summit 2019

ANALYTICAL METHODS

Fluorescence detection at 350 nm and 330 nm→ measures unfolding profile

Temperature Ramp: 20 to 95°C at 1.5°C per min

❑ NanoDSF Simultaneous detection

Light scattering → measures relative amount of aggregation

❑ SEC-HPLC

Equipment: Waters Acquity Arc HPLC/PDA detector/ Empower 3

Column: Xbridge® BEH200Å SEC 3.5μm 7.8 x 300mm

Mobile phase: 25 mM sodium phosphate, 150 mM NaCl, pH 6.8

Running condition:

Isocratic, Wavelength 218 nm 0.5 mL/min for 30 min

Injection volume: 10 µL

Prometheus NT.48

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▪ Significant ↓ in aggregation at high KLEPTOSE®HP& HPB concentration

50

100

150

200

250

300

350

400

450

20 40 60 80 100Bac

k Sc

atte

rin

g (m

AU

)

Temperature (°C)63.0°C

64.0°C

65.0°C

66.0°C

67.0°C

68.0°C

69.0°C

Buffer only 0.05 %Tween 80

5 mMHPBCD

20 mMHPBCD

100 mMHPBCD

T_A

gg (

°C)

Aggregation Onset Temperature (°C)KLEPTOSE HP

KELPTOSE HPB

Buffer, Tween 80, 5mM, 20 mM

100mM

Aggregation profiles of protein in the presence of KLEPTOSE® HP & HPB

NanoDSF RESULTS

50

100

150

200

250

300

350

400

450

20.0 40.0 60.0 80.0

Bac

k Sc

atte

rin

g (m

AU

)

Temperature (°C)

Controls, 5mM, 20 mM

100 mM

❑ Bevacizumab

❑ Human plasma IgG

Aggregation profiles

65.0

66.0

67.0

68.0

69.0

70.0

71.0

CF CF w/oTween 20

5mMHPBCD

20mMHPBCD

100mMHPBCD

T_A

gg.(

°C

)

Aggregation Onset Temperature (°C)

KLEPTOSE HP

KLEPTOSE HPB

1712 Mar 2019 Global Delivery and Formulation Summit 2019

NANODSF RESULTS

Thermal unfolding of IgG

0.740

0.745

0.750

0.755

0.760

0.765

0.770

Bufferonly

0.05 %Tween

80

5 mMHP

20 mMHP

100 mMHP

5 mMHPB

20 mMHPB

100 mMHPB

F35

0/3

30

@ 2

5°C

T0

96 hShaking

0.75

0.77

0.80

0.82

0.85

0.87

0.90

0.92

0.95

20 30 40 50 60 70 80 90

F35

0/3

30

Temperature (°C)

% unfolded

IgG Thermal unfolding curve

• Lower % unfolding observed in formulations containing KLEPTOSE HP/HPB

• KLEPTOSE HP/HPB prevent unfolding of IgG from agitation stress

❑ Effect of agitation stress

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HUMAN PLASMA IgGAgitation Stress

❑Effect of KLEPTOSE HP/HPB on agitation-induced aggregation

• % soluble monomer was higher in formulations with KLEPTOSE® HP/HPB compared to control or Tween 80

• High concentration of KLEPTOSE® HP/HPB (i.e. 100 mM) was able reduce the rate of aggregation

11.5

12

12.5

13

Frag

men

t (%

)

% FragmentsT0

Shake 48 h

Shake 96 h

1.5

2.0

2.5

3.0

3.5

4.0

Solu

ble

Agg

rega

te (

%)

% Soluble aggregates

T0

Shake 48 h

Shake 96 h

• Longer duration of agitation resulted in fragmentation of monomer, which could be inhibited by KLEPTOSE® HP/HPB

Buffer

only

0.0

5%

Tw

een-8

0

5 m

M H

P

20 m

M H

P

100 m

M H

P

5 m

M H

PB

20 m

M H

PB

100 m

M H

PB

8 08 59 09 5

9 5

9 6

9 7

9 8

9 9

1 0 0

% S o lu b le m o n o m e r

T0

S h a k e 4 8 h

S h a k e 9 6 h

So

lub

le m

on

om

er

(%)

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BEVACIZUMABAgitation Stress

❑ Effect of KLEPTOSE® HP/HPB on agitation-induced aggregation

➢ Bevacizumab is stable towards agitation. Minimal increase in protein aggregation was observed after extensive shaking.

➢ However, it was observed that the rate of aggregation was lower in formulations containing KLEPTOSE®HP& HPB as compared to the commercial formulation.

➢ KLEPTOSE ® HP& HPB may possibly serve as an alternative to polysorbates in bevacizumab formulations

1.8

2.0

2.2

2.4

2.6

2.8

CF CF w/oTween

20

5 mMHP

20 mMHP

100 mMHP

5 mMHPB

20 mMHPB

100 mMHPB

Solu

ble

Agg

rega

te (

%)

Soluble Aggregate (%)

T0

24 h Shaking

96 h Shaking

2012 Mar 2019 Global Delivery and Formulation Summit 2019

HUMAN PLASMA IgG

Heat Stress

❑Effect of KLEPTOSE HP/HPB on heat-induced aggregation

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

% S

olu

ble

agg

rega

te

Soluble Aggregate (%)

T0

1 week @40 deg C

+1.58 +1.67 +1.21 +1.36 +1.23 +0.82 +1.25 +1.13 +0.75

➢ KLEPTOSE® HP and HPB reduced heat-induced aggregation in a concentration-dependent manner.

➢ KLEPTOSE® HP and HPB when used at optimized concentrations can be as effective as trehalose in inhibiting protein aggregation.

Note: Numbers above chart represent the increase in soluble aggregate after heat stress

2112 Mar 2019 Global Delivery and Formulation Summit 2019

REPRESENTATIVE SEC CHROMATOGRAMS

Human plasma IgG

➢ Human plasma IgG in buffer before and after 1 week heat stress at 40 °C

Aggregate

Monomer ----- T0----- 1 week @ 40 °C

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1.80

2.00

2.20

2.40

2.60

CF CF w/oTween 20

5 mMHPBCD

20 mMHPBCD

100 mMHPBCD

Solu

ble

Agg

rega

te (

%)

soluble aggregate (%)KLEPTOSE HP

KLEPTOSE HPB

1.00

1.50

2.00

2.50

3.00

3.50

Buffer only 0.05%Tween-80

5 mMHPBCD

20 mMHPBCD

100 mMHPBCD

% S

olu

ble

Agg

rega

te

soluble aggregate (%)

KLEPTOSE HP

KLEPTOSE HPB

REVERSIBLE AGGREGATE REDUCTION

➢ Soluble aggregate (%) decreased with addition of KLEPTOSE® HP/HPB➢ Hypothesis: KLEPTOSE® HP & HPB may have the ability to interfere with the reversible self-

association of protein monomers. ➢ Hypothesis requires additional studies to confirm.

❑ Evaluation of reversible aggregate reduction property of KLEPTOSE® HP & HPB

➢ KLEPTOSE HP& HPB were added into unstressed formulations of human plasma IgG and bevacizumab at three different concentrations

➢ % soluble aggregate was measured using SEC .

❑ Human plasma IgG ❑ Bevacizumab

2323

GENERAL CONCLUSIONS

Case Study

➢ KLEPTOSE® HPB & HP are surface active, may serve as alternative surfactants in protein formulations

➢ Anti-aggregation properties of KLEPTOSE® HP & HPB confirmed using orthogonal methods (nanoDSF & SEC):

➢ IgG

❑Reduce rate of aggregation during agitation and heat stress

❑Prevent fragmentation during agitation

❑Reduce protein unfolding

➢Bevacizumab

❑Reduce rate of aggregation during agitation

❖ KLEPTOSE® HPB & HP are promising excipients that can reduce therapeutic protein

aggregation induced by physical stresses, minimizing protein and efficacy loss

❖ KLEPTOSE® HPB & HP can serve as an alternative option to presently used excipients,

or as a tool to synergistically complement them in biologic formulations

2412 Mar 2019 Global Delivery and Formulation Summit 2019

THANK YOU