The invention relates to a white, flame-resistant, UV-stable, thermoformable, oriented film made from a crystallizable thermoplastic, the thickness of which lies in the range of from 10 xcexcm to 350 xcexcm. Said film comprises at least one white pigment, a flame-proofing agent and a UV absorber and is characterized by good stretchability and thermoformability, by good optical and mechanical properties and an economical production. The invention further relates to a method for the production of said film and the use thereof.

The invention relates to a white, flame-retardant, UV-resistant, thermoformable, oriented film made from a crystallizable thermoplastic, the thickness of the film being in the range from 10 to 350 xcexcm. The film comprises at least one white pigment and one flame retardant and one UV absorber and has good orientability and thermoformability, and very good optical and mechanical properties, and can be produced cost-effectively. The invention further relates to the use of this film and to a process for its production.
White, oriented films made from crystallizable thermoplastics with a thickness of from 10 to 350 xcexcm are well known.
These films do not comprise UV absorbers of any kind as light stabilizers and do not comprise flame retardants of any kind, and therefore neither the films nor the items produced from them are suitable for outdoor applications which demand fire protection or flame retardancy. The films do not pass the fire tests to DIN 4102 Part 2 and Part 1, or the UL 94 test. The films have inadequate thermoformability.
Even after a short time in outdoor applications, these films yellow and exhibit impairment of mechanical properties due to photooxidative degradation by sunlight.
EP-A-0 620 245 describes films with improved heat resistance. These films comprise antioxidants which are suitable for scavenging free radicals formed in the film and degrading any peroxide formed. However, that specification gives no proposal as to how the UV resistance of these films might be improved.
DE-A 2346 787 describes a flame-retardant polymer. Alongside the polymer, the use of the polymer is also claimed for producing films or fibers.
The following shortcomings were apparent during production of films from this phospholane-modified polymer:
The polymer is very susceptible to hydrolysis and has to be very thoroughly predried. The polymer cakes during its drying by prior-art dryers, and it is impossible to produce a film except under the most difficult of conditions.
The films produced under extreme and uneconomic conditions embrittle on exposure to heat, i.e. the mechanical properties are severely impaired due to substantial embrittlement, making the film unusable. This embrittlement occurs after as little as 48 hours of exposure to heat.
It was an object of the present invention to provide a white, flame-retardant, UV-resistant, thermoformable, oriented film with a thickness of from 10-350 xcexcm which not only can be produced cost-effectively and has good orientability and good mechanical and optical properties, but in particular is flame retardant, does not embrittle on exposure to heat, is thermoformable, and has high UV resistance.
Flame retardancy means that in a fire test the white film complies with the conditions of DIN 4102 Part 2 and in particular the conditions of DIN 4102 Part 1, and can be allocated to construction materials class B 2 and in particular B1 for low-flammability materials.
The film is also intended to pass the UL 94 test xe2x80x9cVertical Burning Test for Flammability of Plastic Materialxe2x80x9d, permitting its classification in class 94 VTM-0. This means that 10 seconds after removal of the Bunsen burner the film has ceased to burn, and after 30 seconds no glowing is observed, and no drips are found to occur.
High UV resistance means that sunlight or other UV radiation causes no, or only extremely little, damage to the films, so that the films are suitable for outdoor applications and/or critical indoor applications. In particular, after a number of years in outdoor applications the films are intended not to yellow, nor to exhibit any embrittlement or surface cracking, nor to exhibit any impairment of mechanical properties. High UV resistance therefore means that the film absorbs UV light and does not transmit light until the visible region has been reached.
Thermoformability means that the film can be thermoformed to give complex and large-surface-area moldings on commercially available thermoforming machinery, without uneconomic predrying.
Examples of good optical properties include uniform coloration, high surface gloss ( greater than 15), low light transmission ( less than 70%), and also a Yellowness Index unchanged from that of the flame-retardant and UV-modified film.
Good mechanical properties include high modulus of elasticity (EMD greater than 3200 N/mm2: ETD greater than 3500 N/mm2), and also good values for tensile stress at break (in MD  greater than 100 N/mm2; in TD  greater than 130 N/mm2).
Good orientability includes the capability of the film to give excellent orientation, both in a longitudinal direction and I transverse direction during its production, without break-offs.
Cost-effective production includes the capability of the raw materials or raw material components needed to produce the flame-retardant film to be dried using industrial-standard dryers. It is important that the raw materials neither cake nor become thermally degraded. These prior-art industrial dryers include vacuum dryers, fluidized-bed dryers, fixed-bed dryers (tower dryers). These dryers operate at temperatures of from 100 to 170xc2x0 C., at which the flame-retardent polymers cake and have to be dug out, making film production impossible.
In the case of the vacuum dryer, which provides the mildest drying conditions, the raw material traverses a temperature range from about 30 to 130xc2x0 C., under a vacuum of 50 mbar. Post-drying is then needed in a hopper at temperatures from 100 to 130xc2x0 C. with a residence time of from 3 to 6 hours. Here, too, this polymer cakes to an extreme extent.
This object is achieved by means of a white thermoformable film with a thickness in the range from 10 to 350 xcexcm, which comprises a crystallizable thermoplastic principal constituent, and comprises at least one white pigment, at least one UV absorber, and at least one flame retardant, where expediently the UV absorber and according to invention the flame retardant are fed directly as masterbatch during the production of the film.
The white, flame-retardant, UV-resistant, thermoformable, oriented film comprises, as principal constituent, a crystallizable thermoplastic. Examples of suitable crystallizable or semicrystalline thermoplastics are polyethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, preferably polyethylene terephthalate.
According to the invention, crystallizable thermoplastics are crystallizable homopolymers, crystallizable copolymers, crystallizable compounded materials (mixtures), crystallizable recycled material, and other types of crystallizable thermoplastics.
The white film may be either a single-layer or a multilayer film. The film may also have a coating of various copolyesters or adhesion promoters.
According to the invention, the white film comprises a UV absorber and a flame retardant. The UV absorber is expediently fed directly during the production of the film by way of masterbatch technology, the concentration of the UV stabilizer preferably being from 0.01 to 5% by weight, based on the weight of the layer of the crystallizable thermoplastic.
No embrittlement on brief exposure to heat means that after 100 hours of a heat-conditioning procedure at 100xc2x0 C. in a circulating-air oven the film or the molding exhibits no embrittlement nor any poor mechanical properties.
The film of the invention comprises at least one flame retardant, fed directly during the production of the film by way of masterbatch technology, the concentration of the flame retardant being in the range from 0.5 to 30.0% by weight, preferably from 1.0 to 20.0% by weight, based on the weight of the layer of the crystallizable thermoplastic. The ratio of flame retardant to thermoplastic maintained during production of the masterbatch is generally in the range from 60:40% by weight to 10:90% by weight.
Typical flame retardants include bromine compounds, chloroparaffins, and 10 other chlorine compounds, antimony trioxide, aluminum trihydrates, the halogen compounds being disadvantageous due to the halogen-containing by-products produced. Another extreme disadvantage is the low lightfastness of a film modified therewith, alongside the evolution of hydrogen halides in the event of a fire.
Examples of suitable flame retardants used according to the invention are organophosphorus compounds, such as carboxyphosphinic acids, anhydrides of these, and dimethyl methylphosphonate. It is important for the invention that the organophosphorus compound is soluble in the thermoplastic, since otherwise the optical properties required are not complied with.
Since the flame retardants generally have some susceptibility to hydrolysis, it can be advisable to add a hydrolysis stabilizer.
Hydrolysis stabilizers used are generally phenolic stabilizers, alkali metal/alkaline earth metal stearates, and/or alkali metal/alkaline earth metal carbonates, in amounts of from 0.01 to 1.0% by weight. It is preferable to use amounts of from 0.05 to 0.6% by weight, in particular from 0.15 to 0.3% by weight, of phenolic stabilizers having a molar mass above 500 g/mol. Particularly advantageous compounds are pentaerythrityl tetrakis-3-(3,5-di-tert-butyl-4-hydroxphenyl) propionate or 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene.
The white pigment is preferably fed by way of masterbatch technology, but may also be incorporated directly at the premises of the polymer producer. The concentration of the white pigment is from 0.2 to 40% by weight, preferably from 0.5 to 25% by weight, based on the weight of the crystallizable thermoplastic.
Preferred suitable white pigments are titanium dioxide, barium sulfate, calcium carbonate, kaolin, silicon dioxide, preferably titanium dioxide and barium sulfate.
The titanium dioxide particles may be composed of anatase or rutile, preferably predominantly of rutile, which has higher opacifying power than anatase.
In a preferred embodiment, the titanium dioxide particles are composed of at least 95% by weight of rutile. They may be prepared by a conventional process, e.g.: by the chloride process or the sulfate process. The amount of these in the base layer is from 0.3 to 25% by weight, based on the base layer, and the average particle size is relatively small, preferably in the range from 0.10 to 0.30 xcexcm.
Titanium dioxide of the type described does not produce any vacuols within the polymer matrix during the production of the film.
The titanium dioxide particles may have the type of covering usually used as a covering for TiO2 white pigment in papers or paints to improve lightfastness, made from inorganic oxides.
TiO2 is known to be photoactive. On exposure to UV radiation, free radicals form on the surface of the particles. These free radicals can migrate into the film-forming polymers, causing degradation reactions and yellowing.
Particularly suitable oxides include the oxides of aluminum, silicon, zinc, or magnesium, and mixtures made from two or more of these compounds. TiO2 particles with a covering made from two or more of these compounds are described by way of example in EP-A-0 044 515 and EP-A-0 078 633. The coating may also comprise organic compounds having polar and non-polar groups. The organic compounds have to have adequate thermal stability during production of the film by extrusion of the polymer melt. Examples of polar groups are xe2x80x94OH, xe2x80x94OR, xe2x80x94COOX (Xxe2x95x90R, H, or Na, R=alkyl having from 1 to 34 carbon atoms). Preferred organic compounds are alkanols and fatty acids having from 8 to 30 carbon atoms in the alkyl group, in particular fatty acids and primary n-alkanols having from 12 to 24 carbon atoms, and also polydiorganosiloxanes and/or polyorganohydrosiloxanes, e.g. polydimethylsiloxane and polymethylhydrosiloxane.
The coating for the titanium dioxide particles is usually composed of from 1 to 12 g, in particular from 2 to 6 g, of inorganic oxides, and from 0.5 to 3 g, in particular from 0.7 to 1.5 g, of organic compounds, based on 100 g of titanium dioxide particles. The covering is applied to the particles in aqueous suspension. The inorganic oxides may be precipitated from water-soluble compounds, e.g. alkali metal nitrate, in particular sodium nitrate, sodium silicate (waterglass), or silica, in the aqueous suspension.
For the purposes of the present invention, inorganic oxides, such as Al2O3 or SiO2, also include the hydroxides and their various stages of dehydration, e.g. oxide hydrate, the precise composition and structure of which is not known. The oxide hydrates, e.g. of aluminum and/or of silicon, are precipitated onto the calcined and ground TiO2 pigment, in aqueous suspension, and the pigments are then washed and dried. This precipitation may therefore take place directly in a suspension such as that produced within the production process after calcination followed by wet-grinding. The oxides and/or oxide hydrates of the respective metals are precipitated from the water-soluble metal salts within the known pH range: for example, for aluminum use is made of aluminum sulfate in aqueous solution (pH below 4), and the oxide hydrate is precipitated within the pH range from 5 to 9, preferably from 7 to 8.5, by addition of aqueous ammonia solution or sodium hydroxide solution. If the starting material is waterglass solution or alkali metal aluminate solution, the pH of the initial charge of TiO2 suspension should be within the strongly alkaline range (pH above 8). The precipitation then takes place within the pH range from 5 to 8, by addition of mineral acid, such as sulfuric acid. Once the metal oxides have been precipitated, the stirring of the suspension continues for from 15 min to about 2 h, aging the precipitated layers. The coated product is separated off from the aqueous dispersion, washed, and dried at an elevated temperature, in particular at from 70 to 100xc2x0 C.
Light, in particular the ultraviolet content of solar radiation, i.e. the wavelength region from 280 to 400 nm, induces degradation in thermoplastics, as a result of which their appearance changes due to color change or yellowing, and there is also an adverse effect on mechanical/physical properties.
Inhibition of this photooxidative degradation is of considerable industrial and economic importance, since otherwise there are drastic limitations on the applications of many thermoplastics.
Absorption of UV light by polyethylene terephthalates, for example, starts at below 360 nm, increases markedly below 320 nm, and is very pronounced at below 300 nm. Maximum absorption occurs at from 280 to 300 nm.
In the presence of oxygen it is mainly chain cleavage which occurs, without any crosslinking. The predominant photooxidation products in quantity terms are carbon monoxide, carbon dioxide, and carboxylic acids. Besides the direct photolysis of the ester groups, consideration has to be given to oxidation reactions which likewise form carbon dioxide, via peroxide radicals.
In the photooxidation of polyethylene terephthalates there can also be cleavage of hydrogen at the position xcex1 to the ester groups, giving hydroperoxides and decomposition products of these, and this may be accompanied by chain cleavage (H. Day, D. M. Wiles: J. Appl. Polym. Sci 16, 1972, p. 203).
UV stabilizers, i.e. light stabilizers which are UV absorbers, are chemical compounds which can intervene in the physical and chemical processes of light-induced degradation. Carbon black and other pigments can give some protection from light. However, these substances are unsuitable for transparent films, since they cause discoloration or color change. The only compounds suitable for transparent matt films are organic and organometallic compounds which produce no, or only extremely slight, color or color change in the thermoplastic to be stabilized, i.e. those which are soluble in the thermoplastic.
For the purposes of the present invention, UV stabilizers suitable as light stabilizers are those which absorb at least 70%, preferably 80%, particularly preferably 90%, of the UV light in the wavelength region from 180 to 380 nm, preferably 280 to 350 nm. These are particularly suitable if they are thermally stable in the temperature range from 260 to 300xc2x0 C., i.e. neither decompose nor give rise to release of gases. Examples of UV stabilizers suitable as light stabilizers are 2-hydroxybenzophenones, 2-hydroxybenzotriazoles, organonickel compounds, salicylic esters, cinnamic ester derivatives, resorcinol monobenzoates, oxanilides, hydroxybenzoic esters, and sterically hindered amines and triazines, preference being given to the 2-hydroxybenzotriazoles and the triazines.
The UV stabilizer(s) are preferably present in the outer layer(s). The core layer may also have UV stabilizer, if required.
It was highly surprising that the use of the abovementioned UV stabilizers in films gave the desired result. The skilled worker would probably first have attempted to achieve a certain degree of UV resistance by way of an antioxidant, but would have found that the film rapidly yellows on weathering.
In the knowledge that UV stabilizers absorb UV light and therefore provide protection, the skilled worker would be likely to have used commercially available stabilizers. He would then have observed that
the UV stabilizer has unsatisfactory thermal stability, and at temperatures of from 200 to 240xc2x0 C. decomposes and gives rise to release of gases, and
large amounts (from about 10 to 15% by weight) of the UV stabilizer have to be incorporated in order to absorb the UV light and thus prevent damage to the film.
At these high concentrations it would have been observed that the film is yellow even just after it has been produced, with Yellowness Indices (YI) of around 25. It would also have been observed that the mechanical properties of the film have been adversely affected. Orientation would have produced exceptional problems, such as
break-offs due to unsatisfactory strength, i.e. excessively low modulus of elasticity;
die deposits, causing profile variations;
roller deposits from the UV stabilizer, causing impairment of optical properties (defective adhesion, non-uniform surface);
deposits in stretching frames or heat-setting frames, dropping onto the film.
It was therefore more than surprising that even low concentrations of the UV stabilizer achieve excellent UV protection. It was very surprising that, together with this excellent UV protection,
within the accuracy of measurement, the Yellowness Index of the film is unchanged from that of an unstabilized film;
there are no releases of gases, no die deposits, and no frame condensation, and the film therefore has excellent optical properties and excellent profile and layflat, and
the UV-resistant film has excellent stretchability, and can therefore be produced in a reliable and stable manner on high-speed film lines at speeds of up to 420 m/min.
It was more than surprising that the use of masterbatch technology and of appropriate predrying and/or precrystallization and, where appropriate, use of small amounts of a hydrolysis stabilizer permit the production of a flame-retardant and thermoformable film with the property profile demanded in a cost-effective manner and without caking in the dryer, and that the film does not embrittle on exposure to heat and does not fracture when creased. It was very surprising that together with this excellent result and the required flame retardancy, and the thermoformability and high UV resistance
within the accuracy of measurement, the Yellowness Index of the film is not adversely affected when compared with that of an unstabilized film;
there are no releases of gases, no die deposits, and no frame condensation, and the film therefore has excellent optical properties and excellent profile and layflat, and
the flame-retardant UV-resistant film has excellent stretchability, and can therefore be produced in a reliable and stable manner on high-speed film lines at speeds of up to 420 m/min.
With this, the film is also cost-effective.
It was also surprising that a higher diethylene glycol content and/or polyethylene glycol content and/or IPA content than that of standard thermoplastics permits cost-effective thermoforming of the films on commercially available thermoforming plants, and gives the films capability for excellent reproduction of detail.
It is moreover very surprising that it is also possible to reuse the regrind produced from the films or from the moldings without adversely affecting the Yellowness Index of the film.
In one preferred embodiment, the white, flame-retardant film of the invention comprises, as principal constituent, a crystallizable polyethylene terephthalate having a diethylene glycol content of xe2x89xa71.0% by weight, preferably xe2x89xa71.2% by weight, in particular xe2x89xa71.3% by weight, and/or a polyethylene glycol content (PEG content) of xe2x89xa71.0% by weight, preferably xe2x89xa71.2% by weight, in particular xe2x89xa71.3% by weight, from 1 to 20% by weight of an organic phosphorus compound (dimethyl methylphosphonate) as flame retardant soluble in the polyethylene terephthalate, from 0.01 to 5.0% by weight of a UV absorber selected from the group of the 2-hydroxybenzotriazoles or the triazines and soluble in the PET, and from 0.5 to 25% by weight of titanium dioxide whose preferred particle diameter is from 0.10 to 0.50 xcexcm, preferably a rutile-type titanium dioxide. Instead of titanium dioxide, it is also possible to use barium sulfate whose particle diameter is from 0.20 to 1.20 xcexcm as white pigment, the concentration being from 1.0 to 25% by weight. In one preferred embodiment, it is also possible to use a mixture of these white pigments, or a mixture of one of these white pigments with another white pigment.
In one particularly preferred embodiment, the film of the invention comprises from 0.01 to 5.0% by weight of 2-(4,6-diphenyl-1,3,5-triazin-2-yl)-5-(hexyl) oxyphenol of the formula
or from 0.01 to 5.0% by weight of 2,2xe2x80x2-methylenebis(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethyl-butyl)phenol of the formula
In one preferred embodiment, it is also possible to use a mixture of these two UV stabilizers, or a mixture of at least one of these two UV stabilizers with other UV stabilizers, the total concentration of light stabilizer preferably being from 0.01 to 5.0% by weight, based on the weight of crystallizable polyethylene terephthalate.
In the invention it is important for thermoformability that the crystallizable thermoplastic has a diethylene glycol content (DEG content) of xe2x89xa71.0% by weight, preferably xe2x89xa71.2% by weight, in particular xe2x89xa71.3% by weight, and/or a polyethylene glycol content (PEG content) of xe2x89xa71.0% by weight, preferably xe2x89xa71.2% by weight, in particular xe2x89xa71.3% by weight, and/or an isophthalic acid content (IPA) of from 3 to 10% by weight.
The white, UV-resistant, thermoformable, flame-retardant film has the following property profile:
surface gloss, measured to DIN 67530 (measurement angle 20xc2x0), is greater than 15, preferably greater than 20, and light transmittance L, measured to ASTM D 1003, is less than 70%, preferably less than 60%, measured to ASTM S 1003, this being surprisingly good for the UV resistance achieved in combination with the flame retardancy.
Standard viscosity SV (DCA) of the polyethylene terephthalate, measured in dichloroacetic acid to DIN 53728 is from 600 to 1000, preferably from 700 to 900.
The white polyethylene terephthalate film which comprises at least one organic white pigment, one UV stabilizer, and one flame retardant may be either a single-layer film or a multilayer film.
In the multilayer embodiment, the film is built up from at least one corner layer and from at least one outer layer, preference being given in particular to a three-layer A-B-A or A-B-C structure.
For this embodiment it is important that standard viscosity and DEG content and/or PEG content of the polyethylene terephthalate of the core layer are similar to those of the polyethylene terephthalate of the outer layer(s) adjacent to the core layer.
In a particular embodiment, the outer layers may also be composed of a polyethylene naphthalate homopolymer or of a polyethylene terephthalate-polyethylene naphthalate copolymer, or of a compounded material.
Again in this embodiment, standard viscosity and DEG content and/or PEG content of the thermoplastics of the outer layers are similar to those of the polyethylene terephthalate of the core layer.
In the multilayer embodiment, the UV absorber is preferably present in the outer layers. If required, UV absorber may also have been provided in the core layer.
In the multilayer embodiment, the white pigment and the flame retardant are preferably present in the core layer. However, if required, white pigment and/or flame retardant may also have been provided in the outer layers.
In another embodiment it is also possible for white pigment, flame retardant and UV absorber to be present in the outer layers. If required and if fire protection requirements are stringent, the core layer may also have what is known as a xe2x80x9cbase levelxe2x80x9d of flame retardant.
Unlike in the single-layer embodiment, the concentration of the white pigment here, and of the flame retardant and of the UV stabilizer, is based on the weight in the modified layer. Highly surprisingly, weathering tests to the ISO 4892 test specification using the Atlas C165 Weather Ometer have shown that in order to achieve improved UV resistance for a three-layer film it is fully sufficient for the outer layers of thickness of from 0.5 to 2 xcexcm to be provided with UV stabilizers. Fire tests to DIN 4102 Part 1 and Part 2, and also the UL 94 test have equally surprisingly shown that compliance of the film of the invention with the requirements extends to the range of thickness from 5 to 300 xcexcm.
The flame-retardant, UV-resistant, thermoformable, multilayer films produced using known coextrusion technology are therefore of great economic interest when compared with monofilms provided with UV stabilizers and flame retardants throughout, since markedly less additives are needed for comparable flame retardancy and UV resistance.
At least one side of the film may also have been provided with a scratch-resistant coating, with a copolyester, or with an adhesion promoter.
Weathering tests have shown that even after from 5 to 7 years of outdoor use (extrapolated from the weathering tests) the flame-retardant UV-resistant films of the invention generally exhibit no increased yellowing, no embrittlement, no loss of surface gloss, no surface cracking, and no impairment of mechanical properties.
The results of measurements indicate that the film of the invention or the molding does not embrittle when exposed to heat at 100xc2x0 C. over a prolonged period. This result is attributable to the synergistic action of appropriate precrystallization, predrying, masterbatch technology, and modification with UV stabilizer.
The film can be thermoformed without predrying, and can therefore be used to produce complex moldings.
The thermoforming process generally encompasses the steps of predrying, heating, molding, cooling, demolding, and heat-conditioning. Surprisingly, during the thermoforming process it was found that the films of the invention can be thermoformed without prior predrying. This advantage over thermoformable polycarbonate films or thermoformable polymethacrylate films, which require predrying times of from 10 to 15 hours, at temperatures of from 100 to 120xc2x0 C., depending on thickness, drastically reduces the costs of the forming process.
The following process parameters for the thermoforming process were found:
The film of the invention or the molding produced therefrom can moreover be recycled without difficulty and without pollution of the environment, and without loss of mechanical properties, and is therefore suitable for use as short-lived advertising placards, for example, for the construction of exhibition stands, or for other promotional items where fire protection and thermoformability is desired.
An example of a method for producing the white, flame-retardant, thermoformable, UV-resistant film of the invention is the extrusion process on an extrusion line.
According to the invention, the flame retardant is added by way of masterbatch technology. The flame retardant is fully dispersed in a carrier material. Carrier materials which may be used are the thermoplastic itself, e.g. the polyethylene terephthalate, or else other polymers compatible with the thermoplastic.
According to the invention, the UV stabilizer and the white pigment may be fed before the material leaves the producer of the thermoplastic polymer, or during the production of the film, into the extruder.
DEG content and/or PEG content of the polyethylene terephthalate are set at the premises of the polymer producer during the polycondensation process.
Addition of the white pigment and of the UV stabilizer by way of masterbatch technology is particularly preferred. The UV stabilizer and, respectively, the white pigment is fully dispersed in a solid carrier material. Carrier materials which may be used are the thermoplastic itself, e.g. the polyethylene terephthalate, or else other polymers sufficiently compatible with the thermoplastic.
It is important in masterbatch technology that the grain size and the bulk density of the masterbatch are similar to the grain size and the bulk density of the thermoplastic, thus permitting uniform distribution and, with this, uniform UV resistance.
The polyester films may be produced by known processes from a polyester, where appropriate with other polymers, with the flame retardant, with the white pigment, with the UV absorber, and/or with other conventional additives in conventional amounts from 1.0 to not more than 30% by weight, either in the form of a monofilm or else in the form of multilayer, where appropriate coextruded films with surfaces of identical or different nature, for example pigment being present in one surface but no pigment being present in the other surface. It is also possible for one or both surfaces of the film to be provided with a conventional functional coating by known processes.
It is important for the invention that the masterbatch which comprises the flame retardant and, where appropriate, the hydrolysis stabilizer, is precrystallized or predried. This predrying includes progressive heating of the masterbatch at subatmospheric pressure (from 20 to 80 mbar, preferablyfrom 30 to 60 mbar, in particular from 40 to 50 mbar), with stirring, and, where appropriate, post-drying at a constant elevated temperature, again at subatmospheric pressure. The masterbatch is preferably charged at room temperature from a feed vessel in the desired blend with the polymers of the base and/or outer layers and, where appropriate, with other raw material components, batchwise in a vacuum dryer which during the course of the drying time or residence time traverses a temperature profile from 10 to 160xc2x0 C., preferably from 20 to 150xc2x0 C., in particular from 30 to 130xc2x0 C. During the residence time of about 6 hours, preferably 5 hours, in particular 4 hours, the raw material mixture is stirred at from 10 to 70 rpm, preferably from 15 to 65 rpm, in particular from 20 to 60 rpm. The resultant precrystallized or predried raw material mixture is post-dried for from 2 to 8 hours, preferably from 3 to 7 hours, in particular from 4 to 6 hours, in a downstream vessel, likewise evacuated, at from 90 to 180xc2x0 C., preferably from 100 to 170xc2x0 C., in particular from 110 to 160xc2x0 C.
In the preferred extrusion process for producing the polyester film, the molten polyester material is extruded through a slot die and, in the form of a substantially amorphous prefilm, quenched on a chill roll. This film is then reheated and stretched longitudinally and transversely, or transversely and longitudinally, or longitudinally, transversely, and again and longitudinally and/or transversely. The stretching temperatures are generally from TG+10xc2x0 C. to TG+60xc2x0 C. (TG=glass transition temperature), and the stretching ratio for longitudinal stretching is usually from 2 to 6, in particular from 3 to 4.5, and that for transverse stretching is from 2 to 5, in particular from 3 to 4.5, and that for any second longitudinal or transverse stretching carried out is from 1.1 to 5. The first longitudinal stretching may, where appropriate, take place simultaneously with transverse stretching (simultaneous stretching). Heat-setting of the film then follows with oven temperatures of from 180 to 260xc2x0 C., in particular from 220 to 250xc2x0 C. The film is then cooled and wound.
The surprising combination of exceptional properties gives the film of the invention excellent suitability for a wide variety of applications, for example for interior decoration, for exhibition stands or exhibition requisites, as displays, for placards, for protective glazing of machinery or of vehicles, in the lighting sector, in the fitting-out of shops or of stores, as a promotional item or laminating medium, for greenhouses, for roofing systems, external cladding, protective coverings, applications in the construction sector, and illuminated advertising profiles, blinds, or electrical applications.
Its thermoformability makes the film of the invention suitable for thermoforming desired moldings for indoor or outdoor applications.
The invention is further illustrated below using examples.
The following standards or methods are used here in measuring the individual properties.
DEG Content, PEG Content and IPA Content
DEG, PEG, or IPA content is determined by gas chromatography after dissolving the thermoplastic polymer in cresol.
Surface Gloss
Surface gloss is measured at a measurement angle of 20xc2x0 to DIN 67530.
Light Transmittance
Light transmittance is the ratio of the total transmitted light to the amount of incident light. Light transmittance is measured using the xe2x80x9c(copyright)HAZEGARD plusxe2x80x9d tester to ASTM D 1003.
Haze
Haze is that percentage proportion of transmitted light which deviates by more than 2.5xc2x0 from the average direction of the incident light beam. Clarity is determined at an angle of less than 2.5xc2x0.
Haze is [lacuna] using the xe2x80x9cHAZEGARD plusxe2x80x9d tester to ASTM D 1003.
Surface Defects
Surface defects are determined visually.
Mechanical Properties
Modulus of elasticity and tensile stress at break, and tensile strain at break, are measured longitudinally and transversely to ISO 527-1-2.
SV (DCA), IV (DVA)
Standard viscosity SV (DCA) is measured by a method based on DIN 53726 in dichloroacetic acid.
Intrinsic viscosity (IV) is calculated from standard viscosity as follows
IV (DCA)=6.67xc2x710xe2x88x924SV(DCA)+0.118
Fire Performance
Fire performance is determined to DIN 4102 Part 2, construction materials class B2, and to DIN 4102 Part 1, construction materials class B1, and also to the UL 94 test.
Weathering (Bilateral), UV Resistance
UV resistance is tested as follows to the ISO 4892 test specification:
Yellowness Index
(YI) is the deviation from the colorless condition in the xe2x80x9cyellowxe2x80x9d direction and is measured to DIN 6167. Yellowness indices (YIs)  less than 5 are not visually detectable.
In each case, the examples and comparative examples below use white films of varying thickness, produced on the extrusion line described.
All of the films were weathered bilaterally to ISO 4892 test specification, in each case for 1000 hours per side using the Atlas Ci 65 Weather Ometer from the company Atlas, and then tested for mechanical properties, Yellowness Index (YI), surface defects, light transmission, and gloss.
Fire tests to DIN 4102, Part 2 and Part 1, and the UL 94 test, were carried out on all of the films.

White flame-resistant UV-stable thermoformable film made from a crystallizable thermoplastic, a method for production and the use thereof


Methods of affinity separation wherein the affinity ligand is an immobilized proteinaceous ligand wherein one or more of its asparagine (Asn) residues has been modified. Methods of making a stabilized combinatorial protein by a) modification of Asn residues within a protein molecule to increase stability of the protein in alkaline conditions, and b) randomization of a protein molecule to modify its binding characteristics, and combinatorial proteins wherein in a step separate from the randomization step, the stability of the protein in alkaline conditions has been increased by modifying one or more of its Asn residues.

1. Field of the Invention
2. Discussion of the Related Art
The present invention relates to affinity separation and to ligands for use therein.
Affinity separations generally take place on an affinity chromatography column and take advantage of the potentially highly specific nature of interactions involving biomolecules, especially proteins. Other possible separation methods include membrane filtration, two-phase extraction, fluidised beds, expanded beds and magnetic bead separation (Scopes, R. K. Protein Purification, Principles and Practice, 3rd Ed. ISBN 0-387-94072-3). Interactions include protein-protein, protein-nucleic acid, enzyme-substrate, receptor-ligand (e.g. hormone) protein-carbohydrate and protein-metal interactions. Generally, affinity separation is based on the biologically important binding interactions that occur on protein surfaces, such as that between an enzyme and its substrate, between nucleic acid and a DNA binding protein or between antigen and antibody. Either one of a pair of binding partners can be immobilised, e.g. by covalent bonding to an insoluble matrix in an affinity column in order to assay for or purify its binding partner. The very selective nature of interactions between or involving affinity binding molecules, particularly proteins makes them ideal for purification/separation techniques and for many applications, affinity chromatography involving an immobilised protein ligand is preferred over ion-exchange or gel-filtration chromatography.
If a sample is added to a column which carries immobilised on a solid matrix a specific binding partner to a target molecule in the sample, the target molecule will be retained in the column while the majority of the non-target molecules will simply run through. A solution, typically containing a gradually decreasing pH can be added to the column to wash through firstly any non-specifically bound molecules and finally to elute the target molecule. The target molecule, having the highest affinity for the immobilised ligand, will be the last molecule to be washed off the column and it is therefore the final fractions which will contain the highest concentration of the target molecule and can then be used in a further purification/concentration step or be assayed directly or indirectly for the presence of the target molecule.
Medicine and research in the biochemical and biotechnological fields generally, has created a demand for ever purer samples of organic and biological molecules and thus for strategies which can provide samples of high purity as quickly and cheaply as possible.
As well as satisfying a need to obtain pure samples of proteins and other molecules, affinity separation is important in assaying samples quantitatively and qualitatively for a target molecule. This may be important, for example, when blood or urine samples are being assayed for molecules indicative of a disease such as a metabolic disorder or even the presence of non-naturally occurring substances, narcotics, steroid derivatives etc.
An affinity chromatography column, complete with its solid affinity matrix, can be expensive and clearly it is desirable to be able to re-use the column several times, i.e., to complete a number of runs before it has to be discarded due to reduced ability of the immobilised ligand to bind a target molecule or to a reduced specificity of binding. In order to achieve a set of results which can be directly compared and to clear the column of non-specific and specifically bound molecules before performing another run, it is necessary to clean the column. The recognised standard for cleaning and sanitizing separation media and systems is NaOH, often in combination with NaCl. An applied 0.1-1.0 M NaOH solution is able to remove viruses, bacteria, nucleic acid, proteins, yeasts, endotoxins, prions and other contaminating agents. The NaOH contact time may vary, between 30 minutes and 1 hour is typical, and removal from the system is monitored by simple in-line pH and conductivity measurements.
However, the ability of the separation media to withstand these rather harsh sanitizing conditions depends on the functional groups of the attached ligand (binding partner), attachment chemistries, and the stability of the base matrices to alkaline conditions. Proteins are sensitive to extreme pH, such as is experienced during NaOH cleaning and, generally speaking, this will adversely affect the effectiveness of protein-based affinity media. Thus, although protein based affinity separation has advantages over ion exchange and gel-filtration chromatography due to its good specificity, these other less specific techniques are not adversely affected by standard cleaning methods.
The sensitivity of proteins to alkaline pH is primarily due to deamidation of asparagine and glutamine residues, particularly asparagine residues. Deamidation of asparagine results in the formation, via a cyclic imide intermediate, of isoaspartate and aspartate, usually in the ratio of 3:1 to 4:1. This reaction does take place at physiological pH but is far faster at alkaline pH such as present in a chromatography column which is being cleaned by an NaOH solution. The isoaspartyl form is characterised by an atypical amide bond between the xcex2-carboxyl of aspartate and the xcex1-nitrogen of the C-flanking amino acid. This results in an extra xe2x80x94CH2xe2x80x94 in the backbone of the protein as well as a free xcex1-carboxyl group. Cleavage of the peptide backbone may occur as a result of the deamidation and the protein may lose its activity due to a structural change in the whole protein, or merely a small change in a sensitive region such as the active or binding site. The susceptibility of asparagine residues to deamidation is sequence and conformation dependent, Asn residues at Asn-Gly and Asn-Ser sites being particularly vulnerable.
It is a serious problem when an affinity chromatography column has been set up with a protein immobilised on an insoluble support within it and the necessary cleaning process between runs results in reduced efficacy of the system. As well as hastening the end of the absolute useful life of the column, if successive washes decrease the ability of the immobilised protein to capture its binding partner from a sample, comparisons between runs where the concentration of an analyte in a series of samples is measured become meaningless. There is therefore a need for a method of affinity separation wherein the immobilised protein is less susceptible to standard cleaning methods, particularly to alkaline pH.
Thus, in one aspect, the present invention provides a method of affinity separation wherein the affinity ligand is an immobilised proteinaceous ligand wherein one or more of its asparagine residues has been modified.
The term xe2x80x9cmodifiedxe2x80x9d includes deletion of the asparagine residue or replacement of it with a less alkaline-sensitive amino acid, or wherein the asparagine residue has been modified by substitution (i.e. chemical substitution of one or more groups) or other chemical derivitisation, e.g. by a protecting group. Replacement of one or more asparagine residues with a less alkaline-sensitive amino acid is preferred.
By xe2x80x98affinity separationxe2x80x99 is meant any purification or assaying technique which involves the addition of a sample containing a target analyte to a solid which carries on it a specific binding partner to the analyte.
Gravity or other means allows the sample to pass through or across the solid, and the interaction between the analyte and its specific binding partner immobilised on the solid means that the analyte will be retained on the solid while the rest, or most of the rest, of the sample passes through the system. The separation may conveniently be carried out on an affinity chromatography column. The solid is preferably arranged in a column, so that the sample can be added to the top and the non-target part of the sample runs off. An eluant such as a salt solution or a change of pH can be used to displace the specifically bound analyte which can then be collected in a number of aliquots in a controlled manner.
An xe2x80x9caffinity ligandxe2x80x9d is thus a target-specific binding partner, which can be used in an affinity separation process.
xe2x80x98Analytexe2x80x99 is used to refer to any molecule or fragment of a molecule, proteinaceous or otherwise, in a sample which is capable of binding specifically to the immobilised ligand.
The term xe2x80x98specific binding partnerxe2x80x99 may include a molecule or a group of related molecules and any one of these molecules may also bind xe2x80x98specificallyxe2x80x99 to one or more other molecules. Thus, the term does not imply that any one immobilised ligand or analyte can have only one binding partner, rather that binding is specific to the extent that most other molecules will not bind with the same affinity or with the same stringency, particularly that other non-target molecules in a given sample will have a much lower affinity. Purification by affinity separation is often of the order of several thousand-fold because of the high affinity between biologically specific binding partners.
All types of affinity separation which include an immobilised proteinaceous ligand can be used in the method of the invention, and suitable matrices (i.e. solid support) known for use with particular ligands are known in the art, typically based on chromatography using agarose, polacrylamide, silica, polyvinyl styrene, dextran or other polymers. Any of the solid support known in the art for separation or immobilisation processes may be used, as indeed may any of the methods known in the art for attaching molecules such as affinity ligands to solid supports. The proteinaceous ligands are usually attached to the matrix by a coupling agent such as cyanogen bromide, epichlorohydrin, bisoxirane, divinyl sulfone, carbonyl diimidazol, N-hydroxysuccinimide, tosyl/tresyl chloride, epichlorohydrine, carbodiimide, glutaraldehyde, hydrazine, oxirane and also carboxyl or thiol activated matrices and again such coupling agents and coupling chemistries are well known in the art and widely described in the literature (Jansson, J. C. and Rydxc3xa9n, L. Protein purification, 2nd Ed. pp 375-442, ISBN 0-471-18626-0). Various derivatives of matrices which allow straightforward immobilization include CNBr-activated Sepharose 4B, AH-Sepharose 4B and CH-Sepharose 4B and Epoxy-activated Sepharose 6B (Pharmacia).
The proteinaceous affinity ligand may be a molecule having a protein component, which may function as a xe2x80x9cspecific binding partnerxe2x80x9d as defined above. Thus, glycoproteins or protein-lipid complexes or indeed proteins with prosthetic groups may be used as the affinity ligand which is modified according to the present invention. Protein molecules are, however, preferred. The term xe2x80x9cproteinxe2x80x9d is used herein broadly to include any molecule having a polypeptide or peptide structure. In other words, as used herein, a xe2x80x9cproteinxe2x80x9d is made up of a chain of amino acids, and the term does not imply any particular conformational (i.e. tertiary structure) or other requirement.
Suitable amino acids to replace asparagine include any of the other 19 standard naturally occurring amino acids, although cysteine and glutamine would not be preferred. Preferred substituting amino acids include lysine, aspartic acid and leucine. Non-naturally occurring amino acids and amino acid derivatives which are well known to the man skilled in the art could also be used to replace asparagine residues. Significant improvements in the stability of the immobilised protein and thus the efficacy of the column after cleaning can be observed when just one asparagine is substituted by a less alkaline sensitive residue but preferably 2, 3 or more, or even all of the asparagine residues are substituted.
A xe2x80x9cless alkaline-sensitive amino acidxe2x80x9d is one which is less susceptible to degradation under alkaline conditions than Asn, when compared using techniques, methods and conditions known in the art. Such conditions may, for example, be the column washing conditions discussed above, or any other alkaline conditions used in the art to study protein stability or degradation e.g. deamidation as discussed above. A xe2x80x9cless alkaline-sensitivexe2x80x9d amino acid may thus be any amino acid other than Asn, and more preferably other also than Gln and Cys. Conveniently, alkaline-sensitivity may be compared by replacing a given Asn residue in a protein molecule with a substitute amino acid, or by chemically modifying or derivatising the said Asn residues, and then comparing stability in alkaline conditions (e.g. column wash conditions) with the unmodified protein.
As indicated above, the sensitivity of a particular asparagine residue to deamidation will depend on the configuration of the protein, it being particularly important to replace those residues which are on the surface of the three dimensional structure of the protein and thus particularly exposed to the alkaline conditions. It is the overall stability of the ligand which is of most importance and it is desirable to retain the specificity of the interaction between the immobilised ligand and its binding partner, therefore, preferably it is the asparagine residues not involved in the ligand-analyte interaction which are replaced.
The xe2x80x9casparagine modifiedxe2x80x9d proteinaceous affinity ligand (hereinafter the xe2x80x9cmodified proteinxe2x80x9d) may be made by any method known to the skilled man. Standard techniques for site-directed mutagenesis of nucleic acids are described, for example, in the laboratory manual entitled Molecular Cloning by Sambrook, Fritsch and Maniatis. A preferred technique involves PCR mutagenesis, where primers which incorporate the necessary mis-match base pairs to generate the desired mutations are used in a first round of PCR. In the second run, the fragments from the first run are mixed and the polymerase is able to fill in the strands. The resulting double stranded fragment can be ligated into a plasmid which is then used to transform E. coli. The protein can by synthesised in vitro without using a biological host and in this case non-naturally occurring amino acids can be introduced.
As well as the many general uses of affinity separation indicated previously, of particular interest in the present case is the use of affinity separation with ligands which have been made by randomisation (random mutagenesis) of a particular protein to generate ligands with novel, modified or enhanced binding characteristics. These proteins are referred to as combinatorial proteins. Such a technique typically involves random mutagenesis of a target protein, expression of the full library of these variants, e.g. on the surface of filamentous bacteriophage, followed by selection of a protein exhibiting the desired binding characteristics, this selection typically involving a binding reaction between the variant protein and an immobilised ligand (binding partner) i.e. target molecule for the protein, e.g. target analyte. The mutagenesis is random in that the resulting amino acid encoded by any particular codon is not generally pre-determined but the positions where mutations are to be introduced are generally identified in advance. The mutagenesis may involve amino acid substitution deletion, or addition (e.g. insertion).
The use of an expression system such as surface display on phage provides a crucial link between genotype and phenotype; there is a self-contained unit which can be selected on the basis of its specific binding interactions and which also carries the nucleic acid encoding for the protein responsible for the observed binding characteristics. This enables expression in useful amounts of the protein selected for its binding characteristics, such expression typically taking place in a transformed bacterial host.
The protein, selected by its ability to bind to an immobilised ligand (e.g. a desired target molecule (analyte)), is then itself used in affinity separation (i.e. as the affinity-ligand). It is immobilised and used to purify or assay for a target molecule in a sample, typically the same ligand which was used to select the protein in the first place. In this way, a protein from a library of variants can be selected for its ability to bind e.g. insulin, using a column with insulin immobilised on a matrix therein and the selected protein can then be used to test samples for the presence of insulin.
As well as testing for the presence of a target molecule, the affinity separation methods of the invention provide excellent purification methods, yielding samples of a target molecule having good purity. In particular, the methods of the invention will still produce pure samples after many cycles of the separation system, e.g. after many runs, with washing, of a column. Such samples produced by the methods of the invention constitute further aspects of the invention.
If a protein is stabilised by replacing one or more of its asparagine residues with a less alkaline sensitive amino acid prior to the randomisation and selection steps described above, then its useful life as an affinity ligand will be extended due to its ability to withstand the harsh conditions such as high pH which are experienced when an affinity column is washed between runs.
Therefore, a further aspect of the present invention comprises a combinatorial protein wherein in a step separate from the randomisation step (i.e. the step used to generate the combinatorial protein by randomisation of an xe2x80x9coriginxe2x80x9d or xe2x80x9csourcexe2x80x9d or xe2x80x9cstarting proteinxe2x80x9d), the stability of the protein in alkaline conditions has been increased by modifying one or more of its asparagine residues (preferably by replacing one or more of its asparagine residues with a less alkaline sensitive amino acid).
Nucleic acid molecules encoding such a protein as well as cells expressing the protein constitute further aspects of the present invention.
The randomisation may itself result in substitution of asparagine residues but this aspect of the invention is concerned with proteins which have also been specifically modified to replace one or more asparagine residues in order to increase stability. The protein can be stabilised before or after the randomisation or at the same time but preferably the stabilising modifications will be introduced before the randomisation takes place. If stabilisation is performed before randomisation, it need only be performed once, and by selecting from the library of randomised variants against a number of ligands, several stabilised proteins with different binding characteristics can be obtained. If the stabilisation is performed after selection for a particular binding affinity, then there is a risk that some affinity would be lost as a result of the stabilising substitutions.
Techniques for construction of a combinatorial library of protein molecules and subsequent selection to obtain proteinaceous ligands having desired binding characteristics are known in the art (Nygren, P. and Uhlxc3xa9n, M. Current Opinion in Structural Biology (1997) 7: 463-469). Generally, a protein molecule, perhaps having intrinsic beneficial properties such as temperature or pH insensitivity, is used as a scaffold and a combinatorial library is then constructed via random but targeted amino acid substitutions (or other mutations) of that protein molecule, in order to produce a library of molecules having different binding characteristics. Surface residues are generally targeted for random mutagenesis.
Suitable protein scaffolds may simply be linear peptides but preferably the scaffold will possess a folded three dimensional structure which has the potential for higher affinities and is less susceptible to proteolytic degradation. Rather than designing a scaffold de novo, naturally existing proteins or domains are usually selected for further engineering. For the avoidance of doubt, it is to be noted that throughout this specification the word xe2x80x9cproteinxe2x80x9d is used to refer to whole protein molecules as well as domains or fragments thereof, polypeptides or peptides. The choice of protein scaffold depends on several parameters including an ability to be effectively expressed in a desired host organism e.g E. coli when the randomised protein is to be displayed as a fusion protein with a filamentous phage coat protein. The protein should also comprise sufficiently large regions on its surface which are tolerant to substitution (or insertion or deletion etc.) without losing the overall three dimensional structure. If the library is to be produced synthetically, a small overall size is a prerequisite. Where the selected scaffold protein has a binding function, amino acid residues involved in that interaction may be a target for randomisation. Randomisation may be performed in order to enhance known binding properties or to develop ligands with new specificities.
Suitable scaffold molecules are discussed in Nygren et al. (1997) and include cyclic peptides having 40 or more residues in a constrained sequence, immunoglobulin-like scaffolds including Fv or single-chain (scFv) domains, bacterial receptors such as the 58-residue one-domain Staphylococcal protein A (SPA) analogue Z (the xe2x80x9cZ Domainxe2x80x9d being a derivative of the B domain of SPA), or other domains or analogues of SPA, DNA-binding proteins particularly zinc fingers and protease inhibitors. All these molecules can be stabilised by substituting one or more native asparagine residues with a less alkaline sensitive amino acid before a combinatorial library of the pre-stabilised protein is made.
Of particular interest is the bacterial receptor domain Z (Nord, K., Nilsson, J., Nilsson, B., Uhlxc3xa9n, M. and Nygren, P. Protein Engineering (1995) 8, 6, 601-608). This paper by Nord et al. describes a suitable method of constructing a combinatorial library of protein molecules which can be applied to a range of scaffold molecules. The method described is solid-phase-assisted and based on the stepwise assembly of randomised single-stranded oligonucleotides.
Selection from a generated protein library can be performed in a number of different ways known in the art, including bead immobilised libraries (McBride, J. D., Freeman, N., Domingo, G. J. and Leatherbarrow, R. J., J. Mol. Biol. [1996] 259: 819-827), fusions to DNA-binding proteins (Schatz, P. J., Biotechnology [1993] 11, 1138-1143) and when displayed on bacteria (Lu, Z., Murray, K. S., Van Cleave, V., LaVallie, E. R., St{dot over (a)}hl, M. L. and McCoy, J. M. Biotechnology [1995], 13 366-372) or phage (Clackson, T. and Wells, J. TIBTECH (1994) 12, 173-183) as well as yeast cells, (Boder, E. T. and Wittrup, K. D., Nature Biotechnology (1997) 15, 553-557) and in viral systems (Ernst, W., Grabher, R., Wegner, D., Borth, N., Graussauer, A. and Katinger, H. Nucleic Acids Research (1998) 26, 1718-1723 and Grabher, R., Ernst, W., Doblhoff-Dier, O., Sara, M. and Katinger, H. BioTechniques (1997) 22, 730-735).
International Patent Application, publication No. WO 95/19374 describes the generation of a combinatorial library of Z-variants, see in particular Example 4. This application discusses on page 12 the advantages of the Z domain as an affinity ligand in the purification of recombinant proteins, as it is relatively stable in the harsh environment of an affinity column during cleaning. The present invention offers further benefits in that the Z domain can be stabilised to alkaline pH by replacement of asparagine residues. According to the present invention it is proposed to stabilise the native Z domain before the combinatorial library is prepared.
Thus in a further aspect, the present invention provides a method of preparing a combinatorial library of protein molecules wherein the protein has been rendered less sensitive to alkaline pH by modification of one or mote of its asparagine residues before it is randomised (preferably by replacement of one or more of its asparagine residues).
In a yet further aspect of the present invention is provided a method of phage display wherein a protein expressed on the phage surface has had one or more of its asparagine residues modified (preferably by replacement with a less alkaline sensitive residue) in a step separate to any modifications introduced in order to modify binding characteristics of the protein.
A further aspect of the present invention comprises a method of making a stabilised combinatorial protein comprising the steps of:
a) modification of asparagine residues within a protein molecule to increase stability of the protein in alkaline conditions; and
b) randomisation of the protein molecule to modify its binding characteristics.
Modification in step a) will preferably comprise replacement of asparagine residues with other less alkaline sensitive amino acids, Step a) is preferably carried out before step b).
As well as a protein molecule which has been randomised and stabilised, the present invention also relates to fusion proteins comprising a stabilised part and a randomised part, such proteins being useful in certain applications. In particular, this technique enables the development of a stable framework which can have attached thereto a variable region with specific binding characteristics. The variable region can be prepared by randomisation techniques as discussed above and a protein molecule having desired binding characteristics can be selected from the library of variants, for example by phage display and affinity chromatography. This randomised protein can then be expressed as a fusion protein, e.g. in E. coli, together with a protein molecule which has already been engineered to improve its stability to alkaline conditions by replacing one or more of its asparagine residues with other less alkaline sensitive residues.
The fusion protein can be used as an immobilised ligand in affinity chromatography, giving the benefits of a generally stable molecule provided by the framework part as well as a pre-selected binding affinity provided by the randomised part. Such a system may enable the use of a small protein molecule during the randomisation and selection stage as important protein-like characteristics (as opposed to peptide characteristics) are provided by the stabilised part. The same stabilised part can be used together with a variety of different randomised molecules and vice versa. Suitable protein molecules to perform as the stabilised framework part of the fusion peptides include Albumin Binding Protein (ABD); the bacterial receptor domain Z is a suitable molecule for randomization in this context.
Thus the present invention also provides a fusion protein comprising a first part wherein one or more naturally occurring asparagine residues have been modified (preferably replaced by an amino acid residue less sensitive to high pH) and a second part being a randomised protein molecule selected for its specific binding properties.
Nucleic acid molecules encoding such a protein as well as cells expressing the protein constitute further aspects of the present invention.
A further aspect of the present invention comprises a method of stabilising an affinity ligand by modifying one or more of its asparagine residues (preferably the affinity ligand is stabilised by replacing one or more of its asparagine residues with an amino acid residue less sensitive to alkaline pH).
A still further aspect comprises the use of a protein molecule stabilised by modification of one or more of its asparagine residues in surface display or in affinity chromatography.
The term xe2x80x98surface displayxe2x80x99 refers to the technique involved in selection of a protein from a library of molecules which are presented (displayed) in a manner which enables differentiation between the protein molecules on the basis of their binding characteristics.
Surface display is typically performed on the surface of filamentous bacteriophage (phage display) but display can also be on the surface of bacteria, yeast cells or using viral systems. Any xe2x80x9csurface displayxe2x80x9d technique known or proposed in the art, may be used according to the present invention.
A preferred affinity ligand for use in the methods of the invention is Albumin Binding Protein (ABD), a protein domain with affinity towards human serum albumin (HSA). It is derived from a cell wall anchored bacterial receptor protein from Streptococcus G148. It is of particular use as an affinity ligand for the purification of human serum albumin (HSA). The wild type sequence of this protein has four asparagine residues, enhanced stability is observed when just one of these residues is substituted but preferably all four residues are replaced by less alkaline sensitive residues. The wild type amino acid sequence of ABD is:
LAEAKVLANRELDKYGV-SDYYKNLINNAKTVEGVKALIDEILAALP
the asparagine residues have been indicated in bold. The hyphen simply indicates that other molecules in the same family have an additional amino acid in this position. This sequence excludes the 19 amino acid N-terminal xe2x80x98tailxe2x80x99 shown in FIG. 3. Throughout the text particular amino acids of ABD are identified by their position in the full sequence (including the 19 amino acid xe2x80x98tailxe2x80x99) as presented in FIG. 3. Thus, the first asparagine residue of ABDwt which may be stabilised is in position 28 (Asn28). Stabilised versions of ABD for use in the methods of the invention may incorporate some, all or none of the 19 amino acid N-terminal tail.
Stabilised ABD may, as discussed above, be further subjected to randomisation to create a protein (i.e. a combinatorial protein) having e.g. modified binding characteristics for HSA and/or that is able to bind any target of choice and also retains affinity towards HSA.
The randomisation may involve mutagenesis of the same, or preferably different, residues modified in the stabilisation step. Such derivatives of ABD (e.g. mutants created by random or directed mutagenesis) are also included within the scope of the invention.
Thus a further aspect of the present invention is Albumin Binding Protein (ABD) or fragments or derivatives thereof wherein one or more native asparagine residue have been replaced by a less alkaline sensitive amino acid. Nucleic acid molecules encoding such a protein as well as cells expressing the protein constitute further aspects of the present invention.
In a particularly preferred embodiment Asn28 is replaced by leucine, Asn42 by aspartic acid, Asn45, by aspartic acid and Asn46 by lysine (referred to as ABDmut herein). xe2x80x98ABDmutxe2x80x99 is used to refer to proteins incorporating some, all or none of the 19 amino acid N-terminal tail. Typically, if ABDmut is part of a fusion protein, e.g. with domain Z, then only part or none of the N-terminal tail will be present. Asparagine is the most alkaline sensitive amino acid and so any other amino acid residue could be used to replace it and an increase in stability would be expected. It may be of assistance to compare the sequences of other homologous proteins, which are found in other species or perform the same role, to identify suitable amino acids to be used in the substitution.
In a method suitable for the stabilisation of other affinity ligands, the modifications were introduced by PCR mutagenesis. Firstly mismatched primers are used to introduce the mutations and then the fragments from the first run of PCR are mixed and the polymerase allowed to fill in the strands. The double stranded fragment of ABD incorporating the modifications is cleaved with restriction enzymes and ligated into a plasmid restricted with the same enzymes, which is then used to transform E. coli, the expression system for the protein.
Preferably, the affinity ligand purified (i.e. obtained) according to the methods of the invention retains more than 80%, preferably more than 95%, of its normal binding capacity after 20 rounds of treatment with 0.5M NaOH.

Method of affinity separation and ligands for use therein


A hydraulic tensioner for a drive system, including a flexible, inextensible drive element, has a bi-directional travel limiter associated with a hydraulically biased tensioning cylinder. The travel limiter is bi-directional because it limits not only loading of the tensioning cylinder against a tensioner shoe in a direction tending to tighten a drive element, but also resists movement of the tensioner shoe in a direction tending to allow slack in a drive element.

1. Field of the Invention
The present invention relates to a tensioning device for maintaining desired tightness within a flexible inextensible drive element in a system such as a camshaft drive for an internal combustion engine.
2. Disclosure Information
Devices incorporating flexible inextensible drive elements such as fiber-reinforced belts or metal linked chains have traditionally employed tensioning devices. In order to be effective, such devices must damp excess motion of the chain or belt without imposing unreasonably high hub loads upon either the driving member, such as a crankshaft of an engine, or other driven members such as idlers or camshafts. Unfortunately, if tension is not adequate, the chain or belt may skip a tooth, causing unacceptable engine operation or, in extreme cases, engine damage. If, on the other hand tension within the belt or chain is too great, excessive wear will occur within engine bearings such as crankshaft main bearings or camshaft bearings.
It is known to construct a chain tensioner, for driving a camshaft of an internal combustion engine, with the tensioning force being provided by a piston which is pressurized by engine lubricating oil. Such devices usually employ a controlled leakage factor to exert a force of appropriate magnitude upon a tensioner shoe in the direction tending to tension the chain properly, while damping out excess oscillations of the chain. Such tensioners also typically include a ratcheting device which prevents the tensioner shoe from releasing from the chain beyond a predetermined amount when oil pressure drops off, as when the engine is shut down. Unfortunately, such known devices are prone to overextension of the ratcheting tensioner device during startup operating at lower ambient temperatures, which may cause the ills described above as accompanying excess tension in the chain.
It would be desirable to provide a tensioner which not only prevents excessive falloff in chain tension when the engine is shut down, but which also prevents overtightening or overtensioning of a chain or belt during operation of the engine.

Hydraulic tensioner with overtravel limiter