recombinant human albumin. an effective approach for hard ... · ph scouting - protection against...

Eleonora Cerasoli

American DDF Summit - 2019 Boston

Recombinant human albumin.

An effective approach for

hard-to-stabilise biologics

DDF Summit 2019

Formulation of biologics is not a trivial task

Common challenges

Chemical

degradation

Oxidation

Deamidation

Truncation/degradation

Hydrolysis

.

Physical

degradation

Aggregation/fibrillation

Adsorption/depletion

Unfolding

Solubility/precipitation

~5-15% of all formulae require

“advanced”

formulation solutions

2

DDF Summit 2019

Albumin’s natural stabilization properties enables

formulation of challenging APIs

Human albumin as a multifunctional excipient

3

Dose Control: Amphiphilic properties: readily

absorbs to both hydrophobic and hydrophilic surfaces

through a single layer of recombinant albumin

Antioxidant: free thiol group (C34) protects against

modification through oxidative stress from free

radicals

Stabiliser & Aggregation Inhibitor: Binds stressed

protein - Preferential interaction

Stabiliser & Aggregation Inhibitor: via Preferential

exclusion

DDF Summit 2019

Human albumin is used as a stabiliser in a range of

marketed pharmaceutical formulations

Human albumin as a multifunctional excipient

Product Company Albumin [mg/mL]

Avonex® Interferon β 1a Biogen-Idec 15

Rebif® Interferon β 1a MerckSerono 1-2

Botox® Clostridium botulinum type A toxin Allergan 0.5

Varilrix® Varicella virus vaccine GlaxoSmithKline 12.5

Monoclate-P® Factor VIII CSL Behring 15

FSME-Immun Tick borne Encephalitis Vaccine Baxter 1

ACAM2000® Smallpox Vaccine Sanofi 20

Abraxane® Paclitaxel formulated in albumin

nanoparticlesCelgene 45

Zevalin® Ibritumomab tituxetan Spectrum Pharmaceuticals / Bayer Healthcare 75

4

DDF Summit 2019

Recombumin®

Enhanced performance

No endogenous ligands

Batch-to-batch consistency

✓ Unsurpassed cGMP quality meeting USP-NF

standards

✓ Stable and assured long-term supply from reliable

manufacture

✓ Commercially validated in marketed and late-

stage clinical drug and vaccine candidates

✓ Proven acceptability by leading regulatory

agencies

✓ Expert technical and regulatory support

5

DDF Summit 2019

Mechanisms, kinetics and formulation

approaches

Introduction to aggregation

Native state

Non-native states

• Increase colloidal stability of native conformations

• Raise the stability of native conformations and/or destabilisation of intermediate states

• Reduction of rate formation of reversible aggregate

Aggregation prone early

intermediate

(THERMODYNAMIC STABILITY)

Assembly process dominated by

intermolecular forces (B22)

(COLLOIDAL STABILITY)

Growth - Large oligomers

Structural changes

Nucleation step

6

7DDF Summit 2019

Human serum albumin stabilisation against

aggregation

Multifunctional excipient

• Multifunctional excipient

• Inhibition of aggregation across multiple

stress conditions

• Possibility to bring valuable but unstable drug

candidates to market

Dependence from buffers,

buffer components and

endogenous ligand

10DDF Summit 2019

Example: Ibuprofen binding

Endogenous ligands

Recombumin® Prime

(Formulated with Octanoate)Defatted

Recombumin®

Recombumin® Elite

(Formulated without Octanoate)Serum HSA

DDF Summit 2019

Protection against unwanted derivatization

Multifunctional excipient

Direct effect

Albumin has one natural free thiol group (C34) which is,

by evolution, optimized to react with aggressive oxidizing

species.

• C34 enables albumin to protect biopharmaceuticals by

scavenging against modification through oxidation when

in formulation.

Indirect effect

• Albumin, by stabilizing the folded state, can protect

against chemical degradation

9

OxidationDeamidation

Hydrolysis

Stability

Chemical

10DDF Summit 2019

Albumedix recombinant albumins contain the highest

levels of free thiol

Oxidation

Product Source Free thiol[mol SH/mol albumin]

Recombumin® Recombinant from yeast > 0.9 (*)

rAlbumin yeast Recombinant from yeast 0.69

rAlbumin rice 1 Recombinant from rice 0.62

Plasma HSA 1 Plasma derived from human 0.44

rAlbumin rice 2 Recombinant from rice 0.05

Plasma HSA 2 Plasma derived from human 0.02

The level of free thiol is dependent on the process/ source

(*) at point of production.

DDF Summit 2019

A wide range of application areas

Albumedix recombinant human albumin - Recombumin®

Challenging peptide and protein formulations

Sub-unit vaccines formulations

Down-stream processing of viral vaccines or vectors

Biocompatible surface-coating of medical devices

API Solubilisation

Cell therapy preservation and

formulation

11

Peptide hormone: GlucagonWHO list of essential medicines, increases blood sugar levels in hypoglycaemia

Case Study

Known difficult peptide

✓ Physically unstable: associates and the aggregates are characterised by conformational polymorphism.

✓ Chemically unstable: formation of cleavage products and of post translational modifications (PTMs)

✓ Extremely poor aqueous solubility: soluble at pH > 9 or pH < 3

12

Commercial formulation (Recombinant)

• Lyophilized white powder

• The reconstituted solution contains glucagon as hydrochloride salt at 1 mg/mL (1 unit/mL)

• pH 2.5-3.5 after reconstitution - to be administered immediately after reconstitution!

Non-Commercial Glucagon (Synthesized)

• Effect of synthesis impurities on fibrillation kinetics (*)

(*) N- and C-Terminal Hydrophobic Patches Are Involved in Fibrillation of Glucagon. Jesper Søndergaard Pedersen, Dancho Dikov, and, and Daniel Erik Otzen, Biochemistry 2006 45 (48), 14503-14512

DDF Summit 2019

Glucagon in dual-hormone artificial pancreasNeed of stable liquid formulation

Ideal stability for in-pump use

13

References: Journal of Diabetes Science and Technology 2015, Vol. 9(1) 8–16; Diabetes Technol Ther. 2017 Oct;19(10):589-594

Ideal properties

State Liquid

pH and isotonicity Closer to physiological

Distribution and storage Stability 24 months at 5°C and stability at 25

Pump use – Physical stability 7 days at 37°C

Pump use – Chemical stability 10 or more days > 90% at 37°C (RP-HPLC)

Aggregates of different morphology

OxidationDeamidation

Hydrolysis

14

StabilitySolubility

Chemical

Conformational

RP-HPLCESI-MS

SEC-HPLCDynamic Light ScatteringThT Fluorescence

GlucagonHSQGTFTSDYSKYLDSRRAQDFVQWLMNT

Theoretical pI: 6.75

SEC-HPLC

TSK G3000SWXL column

1 ml/min

0.1M NaCl

pH2.4-2.5

SEC-HPLC: Biophysical Journal 13 Mar 2006, 90(11):4181-4194

Stability at 40°C

pH Scouting:50mM Citrate-Phosphate pH5 - pH6 - pH7

H2O pH2.5

40mM Histidine pH540mM Histidine + Arginine40mM Histidine + Glycine

100mM Phosphate100mM Phosphate + Arginine100mM Phosphate + Glycine

15DDF Summit 2019

Glucagon Case Study

Experimental set-up

Solubility

50mM Citrate-Phosphate pH5

40mM Histidine pH5

100mM Phosphate pH5

H2O pH2.5

DDF Summit 2019

Solubility TestsSEC-HPLC: Glucagon Peak Area

Buffer scouting at pH5

16

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Peak A

rea (

mA

U*m

in)

No AlbuminRecombumin®

10 mg/mLNo Albumin

Recombumin®

10 mg/mLNo Albumin

Recombumin®

10 mg/mLNo Albumin

Recombumin®

10 mg/mL

Glucagon

pH2.550mM Citrate-Phosphate 40mM Histidine 100mM Acetate 100mM Phosphate

DDF Summit 2019

Acid solution- pH2.5Aggregation: DLS

Non-Commercial Glucagon - Stabilisation at 40°C

17

No formation of ThT positive aggregates after 2 and

4 weeks at 40°C

1

1.05

1.1

1.15

1.2

1.25

1 100 10000 1000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

Glucagon + Recombumin pH2.5 - 2 weeks at 40°C

Glucagon pH2.5 -2 weeks at 40°C

0

5

10

15

20

25

30

35

40

45

0.1 1 10 100 1000 10000 100000 1000000

% Inte

nsity

Radius (nm)

Glucagon + Recombumin pH2.5 - 2 weeks at 40°C

Glucagon pH2.5 -2 weeks at 40°C

Peak Radius (nm) %Pd %Intensity %Mass %Number

Peak 1 4.9 26 98 100 100

Peak 2 308 8.6 1.9 0 0

Peak Radius (nm) %Pd %Intensity %Mass %Number

Peak 1 1.2 20.6 19.1 99.9 100

Peak 2 13.3 17.1 6.7 0 0

Peak 3 118 33.7 72.7 0 0

Peak 4 4850 14.8 1.4 0 0

0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 9.25 9.5 9.75 10 10.25 10.5Retention time [min]

3x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

Response [m

AU

]

0.6

53

0.8

67

1.1

60

2.7

80

2.9

66

3.1

33

3.2

99

3.5

26

3.7

93

3.9

39

4.2

33

4.6

93

5.4

12

5.6

86

6.2

39

6.6

72

7.0

52

7.3

39

7.7

79

Glu

cagon

8.2

52

8.6

52

9.0

19

9.4

32

9.6

18

9.9

45

10.2

72

10.5

52

(Alb

um

in)

Shoulder sensitivity

Reset baselineShoulder sensitivity

Threshold

Width

Glucagon

Glu std W32019-02-12 10:48:54+00:00 | DAD1A,Sig=210,4 Ref=off | Glu std W32019-02-12 11-35-48+00-00-r002.dx

0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6Retention time [min]

2x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

Response [m

AU

]

2.7

80

3.5

67

4.7

13

6.2

20

6.4

33

6.6

86

6.8

93

7.0

80

7.3

13

7.9

33

Glu

cagon

8.3

33

8.4

80

9.4

13

9.5

93

9.9

26

10.2

46

10.6

26

(Alb

um

in)

GE control W32019-02-19 10:46:34+00:00 | DAD1A,Sig=210,4 Ref=off | GE control W32019-02-19 10-46-34+00-00.dx

DDF Summit 2019

Acid solution- pH2.5 Chemical degradation: RP-HPLC

Stabilisation at 40°C

18

3 Weeks incubation at 40°C

pH2.4 - no Albumin

3 Weeks incubation at 40°C

pH2.4 + Recombumin®

DDF Summit 2019

pH2.5 and 100mM Phosphate pH5Aggregation: SEC-HPLC


19

0

20

40

60

80

100

120

140

0 12 24 36 48

% R

eco

ve

ry

Time (h)

Glucagon 0.96mg/mL - 100mM Phosphate pH5

Glucagon 0.96mg/mL - 100mM Phosphate pH5, 10mg/mL Recombumin

Glucagon 0.96mg/mL - pH2.5

Glucagon 0.96mg/mL - pH2.5, 10mg/mL Recombumin

pH scouting- Protection against aggregationSEC-HPLC


20

0

20

40

60

80

100

120

0 5 10 15 20 25 30

% R

eco

very

Time (days)

pH7

0

20

40

60

80

100

120

0 5 10 15 20 25 30

% R

eco

very

Time (days)

pH5

0

20

40

60

80

100

120

0 5 10 15 20 25 30

% R

eco

very

Time (days)

pH6

Glucagon Control pH2.4

Glucagon + 20 mg/mL Recombumin® in 50mM Citrate-Phosphate

DDF Summit 2019No formation of ThT positive aggregates

pH2.5 no albumin

pH5 + Recombumin

pH6 + Recombumin

pH7 + Recombumin

DDF Summit 2019

pH scouting - Protection against aggregationDynamic Light Scattering


21

T0

Correlograms

4 Weeks at 40°C

Correlograms

0.98

1.08

1.18

1.28

1.38

1.48

1 10 100 1000 10000 100000100000010000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

0.98

1.08

1.18

1.28

1.38

1.48

1 10 100 1000 10000 100000100000010000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

0

20

40

60

80

100

120

0.1 1 10 100 1000 10000 1000001000000

% I

nte

nsit

y

Radius (nm)

pH5, 6 and 7

Intensity Distributions

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 9.25 9.5 9.75 10 10.25Retention time [min]

3x10

0.2

0.4

0.6

0.8

1

Response [m

AU

]

0.6

53 0.8

60

1.1

53

2.7

67

2.9

40

3.1

33 3.2

87

3.5

13

3.7

80

3.9

27

4.2

27

4.6

80

5.3

87

5.6

47

6.2

00

6.6

46 7.0

26

7.3

20

7.8

00

Glu

cagon

8.2

40

8.6

06

9.0

00

9.4

13

9.6

13

9.9

26

10.2

53

(Alb

um

in)



Threshold

Width

Glucagon

Glu std GE W4 2019-02-20 19:45:55+00:00 | DAD1A,Sig=210,4 Ref=off | Glu std GE W4 2019-02-20 19-45-55+00-00-r001.dx


3x10

0.2

0.4

0.6

0.8

1

Response [m

AU

]

0.6

67

0.7

93

2.8

13

3.2

87

4.2

60

4.6

47

4.8

06

6.1

60

6.9

73

7.2

66

7.5

93

7.7

66

Glu

cagon

8.1

93

8.8

40

9.1

20

9.2

66

9.4

53

9.7

46



Threshold

Width

Glucagon

GPCP5 W4 2019-02-21 00:42:51+00:00 | DAD1A,Sig=210,4 Ref=off | GPCP5 W4 2019-02-21 00-42-51+00-00-r001.dx


3x10

0.2

0.4

0.6

0.8

1

Response [m

AU

]

0.6

60

0.9

00

2.8

13

4.2

66

4.8

06

6.2

79

6.9

73

7.5

93

7.7

46

Glu

cagon

8.1

79

8.8

46

9.1

19

9.2

66

9.4

52

9.7

46



Threshold

Width

Glucagon



3x10

0.2

0.4

0.6

0.8

1

Response [m

AU

]

0.6

60

0.7

13

0.9

93

4.1

40

4.8

07

5.6

13

6.3

20

6.9

73

7.2

47

7.6

13

7.7

73

Glu

cagon

8.1

93

8.3

40

8.6

20

8.8

67

9.1

47

9.4

73

9.7

67

10.1

40

10.3

47



Threshold

Width

Glucagon


DDF Summit 2019

pH scouting - Protection against degradationRP-HPLC


22

pH2.5

50mM Citrate-

Phosphate

+

Recombumin

pH5

pH6

pH7

DDF Summit 2019

pH scouting - Protection against degradationESI-MS


23

Glucagon Control pH 2.4

T0

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

1 10 20

Observed

m/z ratioIdentity

2462P21 Cleavage

(Fragment aa1-21; -1020Da)

FVQWLMNT

Glucagon Control pH 2.4

4 weeks at 40⁰C

1749P15 Cleavage

(F aa1-15; -1734Da)

SRRAQDHSQGTFTSDYSKYLDSRRAQDFVQWLMNT

1 10 20

2522P9 Cleavage

(F aa10-29; -961Da)

Glucagon + Recombumin

in 50mM Citrate-Phosphate pH5

4 Weeks at 40°C

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

1 10 20

DDF Summit 2019

100mM Phosphate buffer pH5 and amino acidsAggregation: SEC-HPLC


24

0

10

20

30

40

50

60

70

80

90

100

110

120

0 24 48 72 96 120

% R

ecovery

Time (hours)

Recombumin + amino acids

Glucagon 0.98mg/mL, 100mM Phosphate pH5

Glucagon 0.98mg/mL, 100mM Phosphate pH5, 20mg/mL Recombumin, 0.11M Arginine

Glucagon 0.98mg/mL, 100mM Phosphate pH5, 20mg/mL Recombumin, 0.27M Glycine

0

5

10

15

20

25

G Phos pH 5 G + Arg 20 mg/mlPhos pH 5

G + Gly 20 mg/mlPhos pH 5

Pea

k A

rea

(m

AU

*min

)

No Albumin Controls

T=0 T= 120h

GlucagonGlucagon +

Arginine

Glucagon +

Glycine

25DDF Summit 2019

100mM Phosphate buffer pH5 + ArginineAggregation: DLS


-1

19

39

59

79

99

119

0.1 1 10 100 1000

% I

nte

nsit

y

Radius (nm)

Intensity Distribution 4 Weeks at 40⁰C

1

1.05

1.1

1.15

1.2

1.25

1.3

1 10 100 1000 10000 100000 100000010000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

Correlograms4 Weeks at 40⁰C

Peak Radius (nm) %Pd %Intensity

Peak 1 3.7 2.52 100

Glucagon Control pH2.4

Glucagon + 10 mg/mL Recombumin® in 100mM PB pH 5

26

40mM Histidine pH5 + Amino acids

Solubility and Stability

-20

80

180

280

380

480

580

680

780

880

0 5 10 15 20 25 30

% incre

ase F

490 n

m

Time (Days)

DDF Summit 2019

40mM Histidine pH5 + Amino acidsAggregation: ThT fluorescence


27

Histidine buffer + Arg or Gly

+ Recombumin®

DDF Summit 2019

40mM Histidine pH5 + Amino acidsAggregation: DLS


28

40mM Histidine pH5, 20 mg/mL Arginine

40mM Histidine pH5, 20 mg/mL Arginine

10mg/mL Prime

1

1.2

1.4

1.6

1.8

2

2.2

1 100 10000 1000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

+ 20 mg/mL Arginine4 Weeks at 40⁰C

1

1.2

1.4

1.6

1.8

2

2.2

1 100 10000 1000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

+ 20 mg/mL Glycine4 Weeks at 40⁰C

40mM Histidine pH5 + Arginine or Glycine

40mM Histidine pH5 + 10 mg/mL Recombumin® + Arginine or Glycine

1

1.2

1.4

1.6

1.8

2

2.2

1 100 10000 1000000

Inte

nsity

Auto

corr

ela

tion

Time (µs)

Only buffer3 Weeks at 40⁰C

0

10

20

30

40

50

60

70

0.1 10 1000 100000

% Inte

nsity

Radius (nm)

Histidine buffer3 Weeks at 40°C

0

10

20

30

40

50

60

70

80

90

100

0.1 10 1000 100000

% Inte

nsity

Radius (nm)

Histidine buffer + Arginine4 Weeks at 40°C

-1

19

39

59

79

99

119

0.1 10 1000 100000

% Inte

nsity

Radius (nm)

Histidine buffer + Glycine4 Weeks at 40°C

29

Accelerated stability studies results

Summary

No Albumin with Recombumin®

SEC-HPLC RP-HPLC ThT Assay DLS SEC-HPLC RP-HPLC ThT Assay DLS

H2O pH2.5 (*) 4 weeks

50mM Citrate-Phosphate pH5 not tested not tested not tested not tested



100mM Phosphate pH5 (**) not tested not tested not tested not tested not tested not tested

100mM Phosphate pH5 + Arginine

100mM Phosphate pH5 + Glycine

40mM Histidine pH5 not tested not tested

40mM Histidine pH5 + Arginine not tested not tested

40mM Histidine pH5 + Glycine

(**) Commercial formulation

DDF Summit 2019

A wide range of application areas

Albumedix recombinant human albumin - Recombumin®

Challenging peptide and protein formulations

Sub-unit vaccines formulations

Down-stream processing of viral vaccines or vectors

Biocompatible surface-coating of medical devices

API Solubilisation

Cell therapy preservation and

formulation

30

Booth N⁰2

recombinant human albumin. an effective approach for hard ... · ph scouting - protection against...

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