Patent Application
20110177955
Proteins have a strong affinity for the surfaces with which they come in contact. In pharmaceutical packages and medical devices this affinity often results in the loss of valuable proteins due to surface adsorption. Surface adsorption is governed by many factors including the nature of a protein, the character of the surface and the additives in the protein solution. Pharmaceutical packagers frequently overfill a container to account for protein loss due to absorption. Many attempts have been made to provide surfaces that resist protein adsorption. No one product can meet the needs of all protein solutions and package performance is highly unpredictable partly because the amount of protein absorption is dependent upon so many factors such as, for example, the pH of the solution, the surface coating and the nature and concentration of the protein. In the past, each new pharmaceutical solution was tested against specific packages or devices and the amount of protein left in the solution was measured to determine the protein loss due to absorption.
Proteins are a heterogeneous class of biomolecules with widely varying physico-chemical characteristics. Some general observations regarding the strength of interaction between proteins and surfaces can be made.
A problem with current pharmaceutical packaging products or medical devices is that no one product possesses each of the positive traits that are needed to provide comprehensive, protein-deterring characteristics. This “mixed bag” of desirable/undesirable characteristics (Table II) renders the performance of products highly unpredictable for universal, protein-based pharmaceutical packaging applications. This unpredictability is evident when considering the highly contradictory results from various “protein adsorption” or “protein loss” studies, which date back to at least 1998. This unpredictability and lack of knowledge about how to truly prevent protein adsorption/loss is ultimately manifested in the inability of the pharmaceutical packaging or medical device industries to develop a single low-loss, protein inhibiting packaging product or device for the pharmaceutical or medical device industry.
The contradictory and sometimes confusing results from previous “protein loss” and “protein adsorption” studies have been further compounded by differences in the testing procedures and assays utilized for assaying “loss” and/or “adsorption”. In previous testing, variability in the testing parameters, including duration of the study, concentration of the protein, testing temperature, pH, use of detergents/additives, etc. have rendered final interpretation and comparison of results difficult or impossible. Further, most “protein loss” assays were conducted using techniques, such as the Bicinchoninic acid (BCA) technique which only allows for the determination of the protein concentration after an adsorption process, but provides no insight into where the proteins were preferentially adsorbed within the pharmaceutical package.
The assay of the present invention will enable drug formulators, medical device developers and pharmaceutical packaging developers to optimize formulations and material surfaces to inhibit the irreversible adsorption of drug compound while utilizing small quantities of compound containing solutions (<<1 ml) and small amounts of potential pharmaceutical packaging or device materials (surface areas <1 mm2). Further, such testing can be achieved in a multiplexed manner (i.e., 2 to 10,000's of formulation/well surface combinations can be assessed on a single, chip-based platform) as shown in
The assay enables a pharmaceutical packager to simultaneously directly compare the adsorption behavior of a specific protein (e.g., recombinant drug, cytokine, enzyme etc.) in a solution containing various specific additives (e.g., buffers etc.) under various specific conditions (e.g., pH, temperature etc.) against a variety of potential substrate surface coatings (e.g., silica, polymer coated glass etc.). The multiplexed assay enables the packagers to simultaneously target the specific packaging conditions (e.g., surface coating, pH, additives) that will result in the least amount of protein adsorption and thus product loss.
Generally, the present invention relates to a multiplexed assay that allows simultaneous measurement of the adsorption interaction of one or more protein solutions with one or more substrate surfaces. Briefly, a substrate is divided into multiple wells, each of which has a surface to be tested, e.g., the substrate surface per se or one which is coated or treated in some fashion. Each well of the multiple well substrates is then subjected to, i.e., exposed to a protein solution and the level of protein adsorption in each of said wells is determined. (The term “adsorption” is not intended to place any limitation on the nature of the interaction between the assayed component of a solution and the test surface. As long as the interaction is sufficient to keep the component in association with the surface sufficiently to be detected in an assay, it is included within the scope of the term.)
Preferably, the substrate is a glass slide or microtiter plate. Each substrate may contain from 2 to greater than 10,000 wells that are created, e.g., with a hydrophobic patterning material. Preferably, the substrate contains greater than 4 wells per substrate. More preferably, the substrate contains greater then 8 wells per substrate. Most preferably, the substrate contains greater then 16 wells per substrate. The protein solutions may contain buffers, salts, stabilizers, preservatives, acids and/or bases, etc., as are common in the pharmaceutical industry. Typically, the protein to be tested is an antibody, an enzyme, recombinant erythropoietin, a recombinant hormone, polypeptides in general, peptides, vaccines, etc. The level of protein adsorption in each of said wells may be determined by, for example, incubating the wells with labeled antibodies and scanning to determine the amount of protein bound. Alternatively, the level of protein adsorption in each of said wells may be determined by, for example, interrogation with an enzyme conjugated antibody and measuring the signal amplification. Thus, the amount of protein that adsorbs to the substrate surfaces under various conditions and various solution parameters can be easily determined.
Various features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:
Surfaces susceptible to protein adsorption include pharmaceutical packaging components (e.g., glass vials, ampoules, stoppers, caps, ready to fill syringes—glass and plastic, cartridge-based syringes, pure silica-surfaced vials, plastic-coated glass vials, plastic and glass storage bottles, pouches, pumps, sprayers and pharmaceutical containers of all types) and medical devices (e.g., catheters, stents, implants, syringes etc). Any candidate surface which is considered for contact with a protein and is susceptible to protein adsorption can be assayed.
Typically, the assay substrate material is glass or plastic. Preferably the substrate material is a SiO2 based glass slide. Most preferably the glass comprises a commercially relevant glass which is used in pharmaceutical packaging applications such as, for example, one comprising 65-85 wt % SiO2, 3-20 wt % B2O3, 0-20 wt % Al2O3, 1-15 wt % Na2O, 1-15 wt % K2O, 0-10 wt % MgO, 0-10 wt % CaO and 0-10 wt % BaO.
A variety of known commercially available patterning compositions such as PTFE polymer (Poly Tetra Flourine Ethylene), fluoropolymers or silicone can be applied to the substrate to create hydrophobic boundary regions thereby creating wells. The boundary regions (i.e., walls) provide a barrier between each well surface (bottom, typically) where assaying reactions can be conducted.
It is contemplated that wells of the patterned substrate can in one embodiment remain uncoated and untreated, with just the underlying substrate composition exposed to the various protein solutions. When the well area has not been additionally coated or treated, then the substrate surface for that well will be an untreated substrate surface. Alternatively, each well can be treated or coated with a different test coating or surface treatment. It is also contemplated that each well can be treated or coated with the same substrate treatment or coating or multiplexed treatments and/or coatings. Or some of the wells may be treated and others may be coated. Obviously a large variety of combinations of well coatings and/or treatments can be tested on a single substrate.
Substrate surfaces comprise uncoated, coated, treated or untreated well surface areas such as, for example:
1) Glass (e.g., silicates, borates, borosilicates, phosphates, etc);
2) Glass that has been heat-treated with an oxy-fuel flame to emulate the processing utilized to convert glass tubing into pharmaceutical packaging;
3) Polymeric materials, such as acrylics, polycarbonates, polyesters, polypropylenes, polyacetals, polystyrenes, polyamides, polyacrylamides, polyimides, polyolefins, cyclic olefin copolymers, especially bicyclic olefin copolymers, or polymeric films;
4) Organic coatings having the following functional group(s) present at the well surface: amine, epoxide, isocyanate, isothiocyanate, acyl azide, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, sulfonyl chloride, imidoester, carbodiimide, acid anhydride, iodoacetyl, malemide, aziridine, acryloyl, disulfide, diazoacetate, aryl azide, thiol (sulfhydryl), mercapto, acetate, hydroxyl, carbonate, aldehyde, alkane, alkene, carboxylate, esters, ethers, etc.—(this is a non-exhaustive list of potential functional groups); organic coatings/films composed of dendrimers, polymers (polyethylene glycols—PEG), nanoparticles, and hyper-branched polymers, e.g., that contain the aforementioned functional groups;
5) Metallic coatings such as gold, silver, platinum, palladium, etc; and/or
6) Inorganic oxide coatings such as SiO2, TiO2, ZrO2, Al2O3, etc.
Many other possibilities exist, e.g., other surfaces used in the pharmaceutical packaging and medical device fields.
Other typical surface treatments or coatings include polymer coated surfaces, native glass, etched glass, thermal treated glass, borosilicate glass coated with a PEG layer, Hyal, siliconized glass or plastic, TopPac, Type 1, Type 1 plus. It is contemplated that any pharmaceutical packaging or medical device surface, surface treatment or coating can be tested against the various parameters of protein solutions. There is a wealth of general knowledge regarding surfaces and or coatings that resist protein adsorption. See, for example, Emanuele Ostuni, Lin Yan, George M. Whitesides—Colloids and Surfaces Biointerfaces 1999, 15, 3-30. Additionally, there is a wealth of general knowledge regarding surfaces that are designed to decrease protein adsorption. See, for example, Emanuele Ostuni, Robert G. Chapman, R. Erik Holmin, Shuichi Takayama, George M. Whitesides—Langmuir 2001, 17, 5605-5620. A large variety of surface coating combinations can exist on a single substrate. All of these represent the large number of available surface treatment and/or coating possibilities.
As used herein, the term “protein solution” refers to a particular protein of interest in the presence of (typically) an aqueous solution that may contain various additives. Typical protein solutions to be tested include pharmaceutically relevant moieties such as cells, tissues, and derivatives thereof. Among the proteins are included any polyaminoacid chain, peptides, protein fragments and different types of proteins (e.g., structural, membrane, enzymes, antigens, monoclonal antibodies; polyclonal antibodies, ligands, receptors) produced naturally or recombinantly, as well as the derivatives of these compounds, etc. Specific protein drugs include antibodies (e.g. Remicade and ReoPro from Centocor; Herceptin from Genentech; Mylotarg from Wyeth, Synagis from MedImmune), enzymes (e.g. Pulmozyme from Genentech; Cerezyme from Genzyme), recombinant hormones (e.g., Protropin from Genentech, Novolin from Zymogenetics, Humulin from Lilly), recombinant interferon (e.g., Actimmune from InterMune Pharmaceutical; Avonex from Biogenldec, Betaseron from Chiron; Infergen from Amgen; Intron A from Schering-Plough; Roferon from Hoffman-La Roche), recombinant blood clotting cascade factors (e.g., TNKase from Genentech; Retavase from Centocor; Refacto from Genetics Institute; Kogenate from Bayer) and recombinant erythropoietin (e.g., Epogen from Amgen; Procrit from J&J), and vaccines (e.g., Engerix-B from GSK; Recombivax HB from Merck & Co.).
Typically drug compounds to be tested will be immersed within an aqueous solution that may contain various additives. Such additives have an influence on the binding of a protein drug to a packaging or device surface. Typical additives include buffers (e.g., phosphate, Tween, citrate, and/or acetate), salts such as sodium chloride at physiological concentrations, stabilizers (e.g., anti-oxidants such as histadine, chelators such as EDTA, human albumin or glycerin etc.), preservatives (e.g., phenol, metacresol, benzyl alcohol etc.), and acid or bases (e.g., citric acid, sodium hydroxide, hydrochloric acid, acetic acid etc) to adjust the pH of the formulation to physiologically safe levels.
The patterned and treated substrate can be used to simultaneously investigate the interaction between multiple relevant protein solution parameters (duration, temperature, concentration, pH, etc.) and a variety of surface coatings/treatments using very small amounts of protein. After the assay is completed, the amount of protein adsorbed on each well of the patterned substrate can be detected using known commercially available detection methods for protein adsorption.
The most common analytical techniques for determining protein adsorption take advantage of the change in optical and/or electrical properties of a surface that has adsorbed proteins. These techniques provide a measurement of the presence/absence of species on a surface. Some techniques allow determination of additional information as to the amount or thickness of adsorbed protein (SPR; ellipsometry; QCM; XPS; radioactive isotopic labeling; solute depletion; fluorescence emission spectroscopy), conformation (ATR FT-IR; Raman scattering; XPS; low angle X-ray reflectivity; scanning force microscopy), or binding energy to the surface (scanning force microscopy). Surface plasmon resonance (SPR) is very sensitive to changes in the index of refraction at and near the surfaces of metal films. SPR can measure the before/during/after protein adsorption to determine kinetic and thermodynamic information regarding the adsorption of proteins. See, for example, Jennifer M. Brockman, Anthony G. Frutos, Robert M. Corn—J. Am. Chem. Soc. 1999, 121, 8044-8051. Ellipsometry can be used to determine if proteins have adsorbed to a surface by measuring the change in the index of refraction before/after protein adsorption to give an experimental thickness of the layer of proteins adsorbed. This detection method is useful if a substrate has a refractive index different from the coating. See, for example, Delana A. Nivens, David W. Conrad—Langmuir 2002, 18, 499-504; M. Mrksich, L. E. Dike, J. Tien, D. E. Ingber, G. M. Whitesides—Exp. Cell Res. 1997, 235, 305-313; and Kevin L. Prime, George M. Whitesides—J. Am. Chem. Soc. 1993, 115, 10714-10721. Quartz crystal microbalance (QCM) measures changes in the fundamental frequency of vibration for a quartz crystal for protein adsorption via the piezoelectric effect, yielding adsorbed protein layer thickness. Surface acoustic wave (SAW) and acoustic plate mode (APM) devices takes advantage of changes in surface acoustic waves (velocity and amplitude) when proteins adsorb to the surface of a crystal modified with electrodes, detecting the presence or absence of protein binding. See, for example, Robert Ros Seigel, Philipp Harder, Reiner Dahint, Michael Grunze, Fabien Josse—Anal. Chem. 1997, 69, 3321-3328). X-ray photoelectron spectroscopy (XPS) uses X-rays to eject electrons from atoms; each atom has different XPS spectrum and allows determination of the number and type of atoms per unit area. XPS can also be used to determine if protein has adsorbed to a surface by measuring the spectrum from a protein adsorbed to a surface vs a non-protein adsorbed surface. Attenuated total internal reflectance fourier transfer infrared (ATR FT-IR) spectroscopy examines the twisting, bending, rotating, and vibrational motions of molecules. The spectra provide information that can be used to determine the presence or absence of a protein and give information regarding its conformation on the surface. Low-angle X-ray reflectometry may be used to determine the variations in electron density at an interface and allows resolution of packing differences in layers. Radioactive isotope labeling can be used to quantify the amount of protein adsorbed by ionization detection (Geiger counter) or liquid scintillation. See, for example, Y. S. Lin, V. Hlady and J. Janatova—Biomaterials, 13, (1992), p. 497. Solute depletion measures the amount of protein in solution before or after exposure to a surface. Scanning force microscopy uses a probe tip with a known position to characterize a surface species. The probe tip may be coated with specific molecules to determine chemical and physical interactions with a surface. See, for example, J. N. Lin, B. Drake, A. S. Lea, P. K. Hansma, and J. D. Andrade—Langmuir, 6, (1990), p. 509. Fluorescence emission spectroscopy measures the inherent fluorescence of a molecule or the fluorescence of a fluorescent label on a molecule. Proteins may be fluorescently labeled and detected using fluorimeters. See, for example, V. Hlady, Applied Spectroscopy 1991, 45, 246 and D. J. Sbrich and R. E. Imhof in Topics in Fluorescence Spectroscopy, J. R. Lakowicz Ed., Plenum, New York, (1991), p. 1. Circular dichroism measures the magnitude of polarized light rotation and detects the presence or absence of proteins. See, for example, C. R. McMillin and A. G. Walton—J. Colloid Interface Sci., 84, (1974), p. 345. Raman scattering is complimentary to infrared and measures the vibrational spectrum of molecules that undergo change in polarizability. It is used to determine the presence or absence of specific molecules/functional groups. See, for example, T. M. Cotton in Surface and Interfacial Aspects of Biomedical Polymers, 2, J. D. Andrade Ed., Plenum Press, New York, (1985), p. 161. In general, this invention is not limited in any way by the nature of the forces holding the protein molecules to the substrates.
Fluorescent detection can be utilized as a direct indication as to the amount of a protein bound to a surface. In this method the protein to be studied is conjugated to a fluorescent dye, such as those typically used in DNA and protein microarrays (e.g., Cy-dyes from GE Healthcare, Alexa-flour dyes from Molecular Probes, or other dyes available commercially and used typically to label proteins (dansy/amide, flouresceine)). These dyes are normally conjugated to the protein through amine-reactive groups (typically, NHS-esters, aldehydes or epoxides) and can be easily detected. The quantitative amount of labeled antibody can be measured after washing via laser scanning, utilizing any one of the various commercial scanners from Axon, Perkin Elmer, Alpha Innotech, Tecan, Agilent, Affymetrix, etc. utilized for microarray analysis (as shown pictorially in
If desired, the multiplexed assay of the present invention can be used in conjunction with other protein loss/adsorption assays such as those described in Table III. Once the multiplexed assay has identified desirable formulation and surface combinations, the following assays can be used to test protein loss within a full scale pharmaceutical package or medical device.
The techniques described above can be used in the approach pictorially described in
Thus, the assay of the present invention is useful in allowing packaging and medical device scientists to study the stability of novel new drug compounds such as, for example, small molecules, antibodies, proteins (natural or recombinant), cytokines, vaccines, under a multitude of different packaging and formulation conditions, while consuming very limited amounts of a precious drug compound. Tens of thousands of formulation/well surface combinations can be assessed on a single, chip-based platform. Thus, one can rapidly identify the optimal combination of material surface and product formulation for a given protein-based pharmaceutical compound. The ability to tailor the surface properties of materials and optimize formulations will reduce or eliminate loss of valuable protein due to surface adsorption and allow easy scale-up from assay into a tangible, scalable prototype or a commercial batch.
Although this application is written primarily in terms of proteins, polypeptides or peptides, it can also be applied to other biomolecules such as nucleic acids, polynucleotides (e.g., DNA, RNA, mRNA, pDNA, etc., oligonucleotides), protein/nucleic acid complexes, etc. by straightforward extension application of the invention to biomolecules is routine. Application of this invention to biomolecules is routine. Assay methods and techniques (reagents, signaling methodology, detection methodology, etc.) are all well known.
By “biological specificity” is meant the normal type of biological lock and key type of bonding which is sufficiently unique to identify a species from all others, e.g., antibody-antigen (protein) interactions, receptor-ligand interactions, highly stringent hybridization, etc. Instead, the surface differences here are designed not to identify proteins but to vary adsorption of a protein entity to a treated surface.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The entire disclosure[s] of all applications cited herein are incorporated by reference herein.
a) Choose a substrate which is ideally in the general shape of a microscope slide (nominally 25×75×1 mm3) or a microtiter plate (substrate within the MTP frame has nominal dimensions of 74×110×1 mm3).
b) Partition off the substrate into individual wells (of any shape) with cross sections that can range from <100 μm to several mm. These wells may be formed by, for example, screen-printing a hydrophobic pattern onto the starting substrate, as described in patent application U.S. Ser. No. 10/778,332 titled “Low Fluorescent, Chemically Durable Patterned Substrates for the Attachment of Biomolecules.” The resulting patterned substrate can take on the general appearance shown by the examples in
c) Interrogate each individual well (
d) Allow solutions to interact with the well surfaces from a time ranging from 1 sec to 12 months or longer, based on the intent of the study; (e.g., some aging studies may require interaction times of >1 year). During aging, a sealed superstructure can be utilized to inhibit evaporation, as shown in
e) Characterize or measure the amount of drug compound that has irreversibly adsorbed to the well surfaces. This can be done by various methods described above, such as, for example:
Two types of assays demonstrate the adsorption of proteins in solution to glass surfaces. The use of glass slides divided into wells with a silicone superstructure allows incubation of 100 μL volumes of protein solution. To detect and quantify the protein bound to the surface fluorescent dyes are used to ensure adequate sensitivity. Two types of assays, a direct and an indirect assay are discussed.
The direct assay is based on protein solutions, where the protein is modified to contain Cyanine dye (Cy3) (see
In the indirect assay (see
A multiplexed assay is used to assess the adsorption of a protein formulated at different pHs. Proteins have an isoelectric point (pI), which is the pH at which the net charge of the protein is zero. At any pH below the pI the protein will be positively charged, while at pH above the pI the net charge will be negative. Meanwhile the zeta potential for glass is negative at any pH above 3, therefore the glass will be negatively charged above that pH.
Human IgG labeled with Cy3 fluorescent dye (Excitation 532 nm, emission: 535 nm) in a 100 mM Phosphate buffer at pH 5, 6, 7, 8, and 9 is formulated. The pI of IgG is 7.8, therefore at most pH's the protein would be positively charged. 100 μL of the protein solution is incubated in wells formed on slides as described for a period of 72 hours. After incubation the slide wells are washed with 100 μL of water for injection (WFI) three times. The slides are then scanned in a laser scanner.
The images shown in
Given the different nature of proteins in general it is to be expected that different proteins will adsorb to a varying degree to the same surface. In this example the adsorption of different proteins all formulated in the same solutions is tested.
Different aspects of protein characteristics in the proteins selected including large (Fibrinogen, molecular weight 340,000) to small (insulin, molecular weight 5600), acidic pI (albumin, pI 5.2) to basic pI (histone, pI 11.5) are covered. All are formulated in a 100 mM phosphate buffer at pH 5, 7, and 9, and incubated as described in the previous example.
The results shown in
The effect of the surface charge will also modify the adsorption of the proteins. As the negative charges on glass tend to attract positively charged proteins. Positively charged surfaces should tend to repel them and attract negatively charged proteins. Applying an aminosilane coating to the surface of the slides tests this theory. The coating results in a surface of packed amino groups that are protonated. The surface is then incubated with both basic (histone) and acidic (albumin) proteins. As can be seen in
The optimization of the formulation of a protein therapeutic can consider many types of buffers at different pH and concentrations. The methods described within are aimed at increasing the throughput with which these variables are tested.
Protein solutions are made with different buffers and incubated in slide wells as describe in Example I. The proteins are then washed and the slides scanned. The results in
In another case the same protein solution is compared in terms of adsorption with and without the presence of a surfactant typically used in the pharmaceutical industry (Tween-20). The results in
This example shows the utility of the methods in deterring protein adsorption, since just two multiplexed experiments can optimize the conditions to reduce the adsorption of the protein by a factor of 10.
In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/010882 | 10/10/2005 | WO | 00 | 9/21/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60617192 | Oct 2004 | US |
