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Recombumin

Recombinant albumin, human, USP-NF


DATA
SHEET
The future of formulation is clear.
Give your biopharmaceuticals superior protection against aggregation,
oxidation and non-specific adsorption
Recombumin protects against aggregation
A merozoite surface protein, MSP-2, was chosen as a Recombumin® protects MSP-2 malarial antigen against aggregation
valid target to investigate the potential for Recombumin 1.0
to reduce protein aggregation due to its tendency to form

Abs 320nm/Aggregation
0.8
amyloid-like fibril aggregates 1, 2.
0.6
Amyloid-like fibrils were measured by the effect on light
scattering at 320nm. 0.4

RESULTS 0.2
A 50% reduction in aggregation at a 1:1 molar ratio of
0
the antigen to Recombumin was observed (Figure 1). 0 9 14 19 24 29
Recombumin (mg/mL)
Aggregation of the antigen was reduced by 80% in the
Figure 1: The effect of Recombumin in protecting a MSP-2 malarial antigen
presence of the highest concentration of Recombumin. (3.5mg/mL) against aggregation in a concentration dependent manner.

Recombumin also suppressed aggregation of the Recombumin® vs other excipients on suppressing aggregation of the
MSP-2 antigen to a greater extent compared to other MSP-2 malarial antigen
0.7
commercially available excipients (Figure 2).
Abs 320nm/Aggregation

0.6
0.5

Recombumin reduced aggregation 0.4


0.3
of the study protein by up to 80% 0.2
0.1
0
1 10 100
Duration (h)
No excipient Glycine(20mg/mL) Recombumin(15mg/mL) Poly 80(8.2mg/mL) Poly 80(8.2mg/mL) PEG(1.0mg/mL)

Figure 2: Suppression of amyloid-like fibril formation in the presence of


Recombumin and other commonly used excipients.

Superior protection against oxidation


Insulin-like growth factor-I (IGF-I) is an important anabolic Recombumin® protects IGF-I against oxidation
growth factor used clinically in several growth and 19

development deficiency conditions. IGF-I is susceptible to 17


% Oxidized IGF-I

oxidation which also promotes subsequent aggregation 3.


15
Pharmaceutically relevant conditions of protein oxidation
were modelled using hydrogen peroxide. 13

RESULTS 11
Oxidation of IGF-I by was significantly reduced by the 0.1 0.5 2.0 5.0 10.0 20.0 untreated
Recombumin (mg/mL)
presence of increasing concentrations of Recombumin
Figure 3: The effect of Recombumin in protecting IGF-I against oxidation in a
(Figure 3). At the highest Recombumin concentration, concentration dependent manner following exposure to H2O2. (Initial
oxidation of IGF-I was reduced by 93%. It is highly likely IGF-I sample contained 11.6% of oxidized form due to storage alone).
that Recombumin exerts this anti-oxidative effect through
Recombumin® vs L-Methionine in protecting IGF-I against oxidation
the presence of the free thiol at the cys34 residue.
20
The ability of Recombumin to act as an antioxidant was
18
compared to the commonly used L-Methionine (Figure
% Oxidized IGF-I

4). The oxidative protection Recombumin provided was 16

achieved at molar concentrations ~13-fold less than that of 14


L-Methionine. 12

Recombumin reduced oxidation of 10


0 500 1000 1500 2000

IGF-1 by up to 93% and was as Concentration (µM)


Recombumin L-Methionine

effective as L-methionine at molar Figure 4: Percentage of oxidized IGF-I with increasing concentration of
Recombumin and L-Methionine following exposure to H2O2.
concentrations approx 13-fold lower Note:10mg/mL Recombumin is equivalent to 150µM.
Non-specific adsorption is substantially reduced with Recombumin
Transforming growth factor beta-3 (TGF-ß3), an active Loss of TGF-ß3 onto plastic and glass container surfaces due to non-
specific binding
pharmaceutical ingredient (API) in scar prevention, is a 120.0
hydrophobic protein and has a significant tendency to
100.0
adsorb to container surfaces 4. The percentage loss of

% Recovery
80.0
TGF-ß3 due to non-specific binding to polypropylene and
glass vial surfaces in the absence (Figure 5) and presence 60.0

(Figure 6) of Recombumin was assessed. 40.0

20.0
RESULTS
Recombumin was found to substantially reduce protein 0.0
0.1 1 10 100
loss due to non-specific binding to the container surface. TGF-ß3 (µg/mL)
PP Container Glass container

In the absence of Recombumin, non-specific binding Figure 5: TGF-ß3 (0.5-60µg/mL) loss on container surfaces due to non-specific
binding in the absence of Recombumin.
of TGF-ß3 increased progressively at concentrations
TGF-ß3 (0.2µg/mL) binding to a plastic PP container in the presence of
up to 60µg/mL and the recovery of the protein was Recombumin
significantly reduced at lower concentrations (Figure 5). 120.0
In the presence of Recombumin the non-specific binding 100.0
of TGF-ß3 to vessel surfaces was minimal with >95%
% Recovery

80.0
recovery achieved using just 0.05mg/mL of Recombumin
60.0
(Figure 6).
40.0
Polysorbate 80 is widely used in the formulation of
20.0
biotherapeutic products to prevent surface adsorption but
0.0
it can be a potential source of peroxides raising concerns 0 0.1 0.2 0.3 0.4 0.5 0.6
about its suitability as a protein stabilizer 5. Recombumin (mg/mL)
Figure 6: The effect of Recombumin in preventing loss of TGF-ß3 to vessel
The benefit of adding Recombumin was compared to surfaces as a result of non-specific adsorption.

that of Polysorbate 80 in preventing non-specific binding TGF-ß3 binding to a plastic container in the presence of Recombumin®
and Polysorbate 80

© Novozymes Biopharma · Customer communication · 2010-01304-01 · January 2010.


of proteins to plastic (Figure 7) and glass (Figure 8) 120
surfaces. Recombumin prevented the non-specific binding
100
of TGF-ß3 at least as well as Polysorbate 80 in plastic
% Recovery

80
containers and significantly better in glass vials.
60
Recombumin is thought to effect reduction in non-specific
40
binding by occupying binding sites on surfaces that would
20
otherwise be taken by the API protein.
0
Recombumin reduced TGF-ß3 0

0.2 0.4 0.6
TGF-ß3 (µg/mL)
0.8 1 1.2

non-specific adsorption to glass Recombumin (0.1 mg/mL) Polysorbate 80 (0.1mg/mL)

Figure 7: The effect of Recombumin compared to Poly 80 in preventing the non-specific


and plastic surfaces at very low binding of TGF-ß3 from 0.2 to 1.0 µg/mL a to plastic polypropylene container.

concentrations and in a manner TGF-ß3 binding to a glass container in the presence of Recombumin®
and Polysorbate 80
superior to Polysorbate 80 100

80

References 60
% Recovery

1 Xuecheng Zhang, Matthew A. Perugini, Shenggen Yao,Christopher G. 40


Adda, Vincent J. Murphy, Andrew Low,Robin F. Anders and Raymond S.
Norton. Solution conformation, backbone dynamics and lipid interactions 20
of the intrinsically unstructured malaria surface protein MSP2. J. Mol. Biol. 0
(2008) 379, 105-121
2 Christopher G. Addaa, Vince J. Murphya, Margaret Sundeb, Lynne J. 0 0.2 0.4 0.6 0.8 1 1.2
Waddingtonc, Jesse Schloegel a, Gert H. Talboa, Kleo Vingasa, Vivian TGF-ß3 (µg/mL)
Kienzlea, Rosella Masciantonioa, Geoffrey J. Howlett d, Anthony N. Recombumin (0.1 mg/mL) Polysorbate 80 (0.1mg/mL)

Hoddere, Michael Foleya and Robin F. Andersa. Plasmodium falciparum Figure 8: The effect of Recombumin compared to Polysorbate 80 in preventing
merozoite surface protein 2 is unstructured and forms amyloid-like fibrils. the non-specific binding of TGF-ß3 to glass vials
Molecular & Biochemical Parasitology 166 (2009) 159-171
3 Fransson J.R.; Oxidation of human insulin-like growth factor I in CONTACT US FOR FURTHER INFORMATION:
formulation studies. J. Pharm. Sci 1997, 86, 1046-1050.
US sales office (Reg. No: 01142208) · One Broadway · 14th Floor
4 Pellaud J., Schote U., Arvinte T. And Seelig J. Confirmation and self
association of human recombinant transforming growth factor-ß3 in Cambridge · MA 02142, USA
aqueous solutions. 1999. J. Biol. Chem. 274, 7699-7704 Tel: +1 617 401 2500 Fax: +1 617 401 2501
5 Kerwin B.A. Polysorbates 20 and 80 used in the formulation of protein
biotherapeutics: structure and degradation pathways. 2008. J. Pharm. Sci. EU sales office (CVR. No: 29603537) · Krogshoejvej 36
97, 2924-2935
2880 Bagsvaerd · Denmark
Recombumin® is a registered trademark and the property of Novozymes Tel: +45 4446 1021 Fax: +45 4446 0104
Biopharma DK A/S (CVR no. 29603537), a wholly owned subsidiary of
Novozymes A/S. E-mail: biopharma@novozymes.com · www.biopharma.novozymes.com