Surfactant-Stabilized Protein Formulations: A Review of Protein

Chapter 12

Downloaded by NORTH CAROLINA STATE UNIV on December 19, 2012 | http://pubs.acs.org Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch012

Surfactant-Stabilized Protein Formulations: A Review of Protein-Surfactant Interactions and Novel Analytical Methodologies 1,3

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1

LaToya S. Jones , Narendra B. Bam , and Theodore W. Randolph 1

Department of Chemical Engineering, Campus Box 424, ECCH 111, University of Colorado, Boulder, CO 80309-0424 SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406

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Nonionic surfactants play an important role in the pharmaceutics industry. They are found in purification steps as well final product formulations. Despite the extensive use o f nonionic surfactants, their properties, roles and mechanisms by which they yield desired effects are not well understood. This paper discusses the characterization o f nonionic surfactants used in pharmaceutics. A review o f the binary surfactant - water system provides an introduction to the difficulties encountered when studying more complex systems. Surfactant behavior under formulation conditions, surfactant binding to pharmaceutical products, the role of surfactants in protein refolding, and the effects of surfactants on accelerated testing of formulations is the focus of this review.

Proteins have become increasingly important as pharmacotherapeutics over the past twenty-five years. Consequently, the various commercial processes required for protein production such as cell culture, fermentation, reactor dynamics and protein purification and recovery have been the subject of intense research, and significant headway has been made in the understanding of the basic principles in the fields. However, it should be recognized that a process for production o f pharmaceutical proteins must also include storage, shipping, and delivery to the patient. T o maintain a biologically active protein during these last three steps, various excipients are commonly added as stabilizers Understanding the interactions o f proteins with these excipients is essential to the rational design of optimal formulations. 'Current address: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262

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© 1997 American Chemical Society

In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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Polymers, polyols, and synthetic surface-active agents, surfactants, including nonionic (1-3) and anionic surfactants (4-8) have been traditionally used in formulations to stabilize proteins and, in the case o f proteins from blood plasma, to act as antiviral agents (9,10). The utility o f surfactants in the biological realm has been formally explored since the late 1930's. It was around this time that Anson, Sreenivasaya, Pirie, and others realized that it was possible to denature proteins using Duponol surfactants (11,12) SDS is a member o f this family, and its early presence is perhaps one o f the reasons for its dominance in surfactant literature. The behavior and characteristics o f this detergent provide insight on the Duponol and other anionic surfactants. However, the surfactants that are used in formulations for their stabilizing properties are normally nonionic Although these surfactants have been demonstrated empirically to stabilize proteins against long term aggregation and denaturation, the exact mechanism o f interaction is often unclear. In the pharmaceutical industry, a major concern is ease o f approval from the regulating body controlling licensing o f drug products. A n attraction o f nonionic surfactants for use in producing, purifying, and stabilizing drugs is that many have already been approved for use internationally in medicinal products. Table I is a list o f a few o f the approved surfactants The acceptance is based largely on the general low toxicity and low reactivity with ionic species exhibited by these excipients (13). Surfactants traditionally have been added to protein formulations to increase the protein solubility and/or stability Recently, pluronic surfactants have been investigated as in vivo sustained released vessels for pharmacotherapeutics (14). Nonionic surfactants are amphipathic molecules containing a bulky polar head group attached to a hydrophobic chain Chemical sketches o f commonly used nonionic surfactants in the pharmaceutical industry are given in Figure 1. It is important to note that many of the structures are characterized by a polydispersity o f the hydrophobic chain lengths within a given surfactant. This affects another property of surfactants, the critical micelle concentration ( C M C ) . Surfactant concentrations near the C M C are typically the initial concentrations used in formulation development because it is in this range that properties such as interfacial tension are affected greatly. Also, several o f the works referenced in this paper about surfactant - protein interactions discuss trends as a function o f surfactant concentrations as related to the CMC

Determining Critical Micelle Concentration (CMC) and Aggregation Numbers of Pure Micelles There is a tendency to think o f the C M C o f a detergent as a constant. However, not only is the C M C dependent on factors such as ionic strength, p H , temperature, surfactant polydispersity. and the presence o f other excipients, but the method for determining the C M C can also affect the value. F o r these reasons, C M C s and corresponding aggregation numbers are best reported as ranges. Before continuing with methods o f determining C M C s and how the above mentioned factors influence the value, a few definitions are necessary. The C M C is defined as the "limiting concentration o f single (monomeric) molecules that can exist in solution" (20). Micelles will be defined as organized aggregates o f amphophilic In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


Tween 40 Tween 60 Tween 80

Tween 80

Tvveen 80 Tween 80

Tween 80 Tween 80

Brij PEG

Polysorbate 40 Polysorbate 60 Polysorbate 80

Polysorbate 80

Polysorbate 80 Polysorbate 80

Polysorbate 80 Polysorbate 80

Cetomacrogol 1000 Polyethylene Glycol

0

Tubersol (Tuberculin purified protein derivative diagnostic antigen) R h o G A M ( R h (D) Immune Globulin) Neupogen (Filgrastim) Activase (Recombinant Alteplase) Koate-HP (Factor VIII) Kogenate (Recombinant Antihemopphilic Factor)

Actimmune (Interferon gamma-lb)

Final Formulation Usage

< 25 ppm 7H 2

2

Polyethylene glycol ether Triton X-100, x = 9 - 10 (average) Triton X-114, x = 7 - 8 (average) Figure 1: interest.

Chemical structures of nonionic surfactants o f pharmaceutical


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T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y

Micelle

Nonpolar Solvent

V i

Nonpolar Solvent

[ 1

Nonpolar S o l v e n t

Reverse Micelle Figure 2: Surfactant Micelle and Reverse Micelle. The hydrophilic group is represented by a circle, whereas the hydrophobic chain is represented by a "squiggle" line



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compounds (21). These "organized aggregates" are oriented such that the hydrophobic portions are in the interior and the hydrophilic parts are exposed to a polar environment. When the hydrophilic portion is in the interior and the hydrophobic portion is exposed to the nonpolar solvent, the structure is termed reverse micelle. Figure 2 is an example o f each. Reverse micelles will not be discussed further in this paper; however, Luisi and Laane provide reviews on this topic (22). Micellization is a positive cooperative process. The micelle formations o f concern here are those which form spontaneously, corresponding to a reduction in Gibbs free energy o f the system Micellization reduces the interfacial energy between the water and the hydrophobic tails of the surfactant because these tails are no longer in the aqueous environment (Figure 2). Upon initial consideration, this spontaneous ordering may seem strange However, for each monomer sequestered as part o f a micelle, water molecules which were originally structured about it are now less ordered. The forces that must be overcome for micelle formation include stearic hindrance o f bulky head groups, the entropy loss o f the surfactant molecule, and for the case o f ionic surfactants, charge repulsion. It has been emphasized that the hydrophobic effects observed are the result o f the strong attractive forces between water molecules and not due to interactions between hydrophobic moieties o f surfactants (21). Techniques which have been used to determine the C M C o f surfactants include: surface tensiometry, refractive index, light scattering intensity, fluorimetry, sedimentation, azo-hydrozone tautomerism, phase selective ac tensammetry, and spin label partitioning (14,15,23-27) In all o f these techniques, the C M C is described as the concentration at which there is an initial bend in an isotherm o f the measured phenomenon versus concentration Immediately, it is apparent that the number and range o f data points can affect the result. Also, each technique is monitoring a different phenomenon in the solution, with some being more sensitive than others to slight changes. For example, using refractive index and light scattering Kameyama and Takagi determined the C M C for octylglucoside ( O G ) in distilled water at 22 °C to be 25.3 m M (24). This result was in agreement with the reported value o f 25 m M for the C M C o f O G at 25 °C determined by surface tensiometry (28). D e Grip and BoveeGeurts used fluorimetry and arrived at a slightly lower C M C o f 23.2 m M at 25 °C (29) . It should be noted that O G , when compared to most nonionic surfactants o f biological interest, has a relatively high C M C Under similar conditions for the above O G studies, Tritons and Tweens have C M C s less than 1 m M and 0.1 m M , respectively (30) . The consequence o f a relatively low C M C combined with a technique in which micellization produces only a slight discontinuity can lead to erroneous results i f care is not taken in the experimental design Too few data points below the C M C coupled with scatter could result in great difficulty in detecting the C M C .

Membrane Solubilization When therapeutic proteins associated with lipids are formulated with surfactants, the likelihood o f lipid membrane solubilization must be considered. Some nonionic In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y

EtYeet of Average H L B on Membrane Solubilization

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cfl

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Surfactant-Stabilized Protein Formulations: A Review of Protein

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