A tape drive controlling device includes a first controlling circuit for driving a take-up reel for a tape on the basis of the result of a comparison between a take-up-side tension measured between the take-up reel and a head and a predetermined tension target value, a second controlling circuit for driving a supply reel for the tape on the basis of the result of a comparison between a supply-side tension measured between the supply reel and the head and a predetermined tension target value, a speed detecting circuit for detecting the travel speed of the tape, and a correcting circuit for correcting at least one of the take-up-side and supply-side tension target values on the basis of the result of a comparison between a detection result provided by the speed detecting circuit and a travel speed target value.

1. Field of the Invention
The present invention relates to a tape drive controlling device suitable for use in a magnetic-tape transporting system of, for example, a video tape recorder (hereinafter referred to as a "VTR").
2. Description of the Related Art
A magnetic recording and reproducing apparatus using a magnetic tape (hereinafter referred to as a "tape") which is one kind of electronic apparatus provided with such a controlling device, particularly a video tape recorder for business use which is called a "VTR", has conventionally been provided with a shuttle function, i.e., the function of feeding the tape at a high speed while reproducing recorded images one after another for the purposes of cueing or the like during editing.
A typical arrangement of the shuttle function is such that when the travel speed of the tape is to be changed, an operator turns a jog dial to change the travel speed of the tape step by step. In this arrangement and in general, in a low-speed range, the tape is fed by the rotation of a capstan with a pinch roller pressed in contact with the capstan, while, in a high-speed range, the tape is fed by reels with the pinch roller away from the capstan.
However, since the conventional example is arranged in the above-described manner, a number of problems arise. For example, when tape feed is to be switched from capstan feed to reel feed or vice versa in a high-speed travel mode, the operation of bringing the pinch roller into or out of contact with the capstan is needed. This operation hinders smooth change of tape travel speeds, and a considerably long time is taken until a desired speed is reached. Particularly during a transition from the reel feed to the capstan feed, the tape is nipped between the pinch roller and the capstan with the result that the tape feed must be temporarily stopped. This leads to the problem that an image reproducing operation is temporarily stopped.
Another problem may occur during the reversal of a tape travel. This problem will be described below with reference to FIG. 1.
In FIG. 1, a drum provided with a recording and reproducing head is denoted by reference numeral 1, a supply reel by reference numeral 2, a take-up reel by reference numeral 3, a magnetic tape by reference numeral 4, tension detectors for detecting the tension of the tape 4 by reference numerals 5 and 6, and a speed detector for detecting the travel speed of the tape 4 by reference numeral 18.
The magnetic tape 4 is fed from the supply reel 2 to the take-up reel 3 during fast forward feed. A supply-reel motor 17 and a take-up-reel motor 14 directly drive the supply reel 2 and the take-up reel 3, respectively.
The tensions of the tape 4 on a supply side and a take-up side are respectively detected by the tension detectors 5 and 6 and supplied to terminals a and b of the selecting switch 7. The selecting switch 7 is interlocked with selecting switches 10 and 11, and when the tape 4 is to be made to travel in the forward direction (when the tape 4 is to be fed from the supply reel 2 and wound around the take-up reel 3), the signal at the terminal a is selected, while when the tape 4 is to be made to travel in the opposite direction, i.e., in the rearward direction, the signal at the terminal b is selected.
The output from the selecting switch 7 is compared with a tension reference value 9 in a subtracter 8, and an error with the tension reference value 9 is supplied to the terminal b of the selecting switch 10 and to the terminal a of the selecting switch 11.
When the tape 4 is to be made to travel in the forward direction, the selecting switch 7 selects and outputs the signal at the terminal a, and the tension error signal from the subtracter 8 is inputted to a loop filter 15. When the tape 4 is to be made to travel in the rearward direction, the selecting switch 7 selects and outputs the signal at the terminal b, and this signal is inputted to a loop filter 12. Each of the loop filters 12 and 15 performs phase compensation and gain compensation to stabilize an associated servo loop. The outputs from the loop filters 12 and 15 are inputted to reel motor drivers 13 and 16, respectively, and the reel motors 14 and 17 are driven by the associated reel motor drivers 13 and 16.
The output from the speed detector 18 is compared with a speed reference value 20 in a subtracter 19, and the obtained speed error is supplied to the terminal a of the selecting switch 10 and to the terminal b of the selecting switch 11.
In the above-described arrangement, the supply reel 2 is controlled so that the tape tension on the supply side reaches the reference tension value, while the take-up reel 3 is controlled so that the tape 4 is made to travel at a reference speed. Accordingly, it is possible to feed the tape 4 at a desired speed by changing the speed reference value.
However, the above-described conventional example has a number of problems. For example, when the direction of tape feed is changed, a tension control loop and a speed control loop are switched between the supply side and the take-up side with the result that an abnormally high or low tension which may damage the tape is instantaneously applied to it. It has, therefore, been difficult to stabilize the tape tension and smoothly change the direction of tape feed.


In one embodiment, a method includes activating a cross-platform DLP policy for enforcement against a plurality of users on each of a plurality of heterogeneous communications platforms. The method further includes monitoring communications of the plurality of users on each of the plurality of communications platforms for violations of the cross-platform DLP policy. The method also includes, responsive to a detected violation of the cross-platform DLP policy by at least one user on at least one communications platform, dynamically acquiring context information for the detected violation using information associated with the detected violation. In addition, the method includes publishing violation information to one or more designated users. The violation information includes at least a portion of the information associated with the detected violation and at least a portion of the context information.

Technical Field
The present invention relates generally to data security and more particularly, but not by way of limitation, to systems and methods for data loss prevention.
History Of Related Art
Enterprise communications systems such as, for example, email systems (e.g., systems based on MICROSOFT EXCHANGE), sometimes provide administrators with certain tools for setting up automated polices. These policies can help ensure that users comply with standard operating policies or other governance-related requirements. The policies are often specified as rules that define what a user or group of users should or should not do on the system. When the system detects a violation of such policies, the violation can be logged. Each policy, however, is generally platform-specific and is thus typically limited in scope to a particular communications system on which the policy is specified and implemented. For example, an organization may be utilizing different solutions for email, instant messaging, social media, content management, etc. To the extent any native policy support is provided, each enterprise communications system may provide incompatible tools, policy formats, and enforcement actions. Many enterprise communications systems provide no native policy support at all.
In addition, existing policies that are specified can be quite rigid. For example, one policy may be to not send large email attachments. The policy may be specified using an arbitrary maximum attachment size such as, for example, ten megabytes. In practice, however, a given user may send numerous email attachments that are slightly under the maximum attachment size. Although such a user may be literally complying with the policy by not sending email attachments larger than ten megabytes, the user may be violating the spirit of the policy. The user's numerous email attachments of just under ten megabytes may cause the very adverse effects that the policy is designed to prevent. Violations of the spirit of a given policy cannot generally be detected.
Other policies sometimes require interpretation in order to determine a violation. For example, a company policy may be to not email credit-card numbers. An email system, for example, may then implement the policy by searching for a string of numbers that conforms to a credit-card number format (e.g., a string of numbers of a particular length). According to this example, however, benign numbers that are the same length as a credit-card number could trigger a false positive. In general, when reporting a violation, there is not sufficient information available to enable adequate identification of false positives.
Moreover, as the value and use of information continues to increase, individuals and businesses seek additional ways to process and store information. One option available to users is information handling systems. An information handling system generally processes, compiles, stores, and/or communicates information or data for business, personal, or other purposes thereby allowing users to take advantage of the value of the information. Because technology and information handling needs and requirements vary between different users or applications, information handling systems may also vary regarding what information is handled, how the information is handled, how much information is processed, stored, or communicated, and how quickly and efficiently the information may be processed, stored, or communicated. The variations in information handling systems allow for information handling systems to be general or configured for a specific user or specific use such as financial transaction processing, airline reservations, enterprise data storage, or global communications. In addition, information handling systems may include a variety of hardware and software components that may be configured to process, store, and communicate information and may include one or more computer systems, data storage systems, and networking systems.

System and method for data loss prevention across heterogeneous communications platforms


Methods for detecting human factor VIII or factor VIII-like polypeptides in and isolating it from solutions such as blood or conditioned media are disclosed, together with reagents suitable for the purpose comprising binding moieties that recognize human factor VIII and/or a factor VIII-like polypeptide and form a binding complex therewith. Preferred polypeptide binding moieties are particularly disclosed.

The present invention relates to the field of protein isolation and purification. Specifically, the present invention relates to the identification, isolation and synthesis of binding molecules that bind to factor VIII and/or factor VIII-like polypeptides. Such binding molecules are useful for the detection and purification of factor VIII and factor VIII-like polypeptides from solutions containing them.
Classical hemophilia A is the result of a chromosome X-linked deficiency of blood plasma coagulation factor VIII and affects almost exclusively males with a frequency of about 1 case per 10,000. The X-chromosome defect is transmitted by female carriers who do not themselves have the disease. Factor VIII is also known as antihemophilic factor (AHF), hemophilic factor A, platelet cofactor, thromboplastinogen, thrombocytolysin, and antihemophilic globulin (AHG). The designation xe2x80x9cfactor VIII:Cxe2x80x9d is used to indicate that it is the compound that affects clotting. Factor VIII is a high molecular weight protein of 280 kDa and is composed of two polypeptide chains of 200 kDa and 80 kDa, respectively. Andersson et al., Proc. Natl. Acad. Sci. U.S.A., 83:2979-2973 (1986). These chains are held together by a metal ion bridge.
The principal symptom of hemophilia A is bleeding without clotting or coagulation. Prior to the discovery that administration of factor VIII concentrates could ease the symptoms of an individual diagnosed with the disease, the average life expectancy of a sufferer was about 20 years.
Until recent years, the major source of factor VIII for therapeutic purposes was normal blood plasma; however factor VIII isolated by this method, while of some use, has several important disadvantages. For instance, factor VIII isolated from blood plasma is fairly impure, typically having a specific activity of less than 2 units factor VIII/mg protein and an overall factor VIII content of less than 1%. Additionally, the purification process is expensive because the starting material, i.e., human plasma, is expensive. Many precautions must also be taken to decrease the risk of transmitting infectious agents to the patient. For example, human immunodeficiency virus (HIV), Hepatitis B virus, Hepatitis C virus and other disease-causing agents are commonly detected in donated blood. Another disadvantage of using factor VIII obtained by this method is that approximately one-tenth of the patients with severe hemophilia A develop antibodies against factor VIII, making the disease difficult to treat.
Research efforts have focused on the development of methods for creating and isolating highly purified, biologically active factor VIII in full-length and derivative forms. Advantages of a highly purified protein include reduced levels of extraneous proteins in the therapeutic mix as well as a decreased likelihood of the presence of infectious agents. A more purified form of factor VIII may also be administered in smaller doses, possibly reducing the risk of developing anti-factor VIII antibodies, as lower doses would be less challenging to the immune system.
Significant steps have been taken toward the recombinant production of factor VIII beginning with the isolation of biologically active factor VIII fragments. See, Andersson et al., U.S. Pat. No. 4,749,780; Andersson et al., U.S. Pat. No. 4,877,614. The gene encoding the full-length human factor VIII protein was isolated by taking advantage of its sequence homology with porcine factor VIII. See, Toole et al., U.S. Pat. No. 4,757,006. Toole et al. also report the expression of human and porcine protein having factor VIII:C procoagulant activity.
However, severe side effects involving the production of anti-factor VIII antibodies still exist with the administration of the protein isolated from both human and non-human sources. Antibodies that react with human factor VIII:C are also known to react, to a certain extent, with factor VIII:C from other species, and porcine factor VIII itself is antigenic in humans. Also, non-hemophiliacs can develop or acquire the disease when their immune systems become sensitized to factor VIII:C.
As a possible solution to this problem, a truncated, lower molecular weight protein exhibiting procoagulant activity has been designed. See, Toole, U.S. Pat. No. 4,868,112. Toole reported an alternative method of treatment with lower molecular weight porcine factor VIII of approximately 2000 amino acids exhibiting similar procoagulant activity as full-length factor VIII. Evidently, the removal of certain amino acids and up to 19 of the 25 possible glycosylation sites, reduced the antigenicity of the protein and thereby the likelihood of developing anti-factor VIII antibodies. However, one difficulty with the development of recombinant factor VIII is achieving production levels in sufficiently high yields.
Recently, deleted factor VIII cDNA molecules coding for recombinant factor VIII derivatives, which were likely to give sufficiently high yields of a biologically active recombinant factor VIII protein for use in an industrial process for a pharmaceutical preparation have been developed. See, Almstedt et al., U.S. Pat. No. 5,661,008. Almstedt et al. designed a modified factor VIII derived from a full-length factor VIII cDNA, that, when expressed in animal cells, produced high levels of a factor VIII-like protein with factor VIII activity. The protein consisted essentially of two polypeptide chains derived from human factor VIII, the chains having molecular weights of 90 kDa and 80 kDa, respectively.
According to the Almstedt et al. process, the factor VIII cDNAs are assembled into transcription units and introduced into a suitable host system for expression. The cell lines can be grown on a large scale in suspension culture or on solid support. The protein produced in the culture medium is then concentrated and purified. The final active protein is made up of amino acids 1 to 743 and 1638 through 2332 of human factor VIII This polypeptide sequence is commercially known as rFVIII-SQ or REFACTO(copyright). See also, Lind et al., Euro. J. Biochem., 232:19-27 (1995). Other forms of truncated FVIII can also be constructed in which the B-domain is generally deleted. In such embodiments, the amino acids of the heavy chain, consisting essentially of amino acids 1 through 740 of human Factor VIII and having a molecular weight of approximately 90 kD are connected to the amino acids of the light chain, consisting essentially of amino acids 1649 to 2332 of human Factor VIII and having a molecular weight of approximately 80 kD. The heavy and light chains may be connected by a linker peptide of from 2 to 15 amino acids, for example a linker comprising lysine or arginine residues, or alternatively, linked by a metal ion bond.
Currently, there is a need in the field for efficient and cost-effective methods for obtaining purified, active factor VIII directly from various solutions such as blood or cell culture supernatants.
The present invention provides new materials and methods for identifying, isolating, and purifying factor VIII and factor VIII-like proteins, including REFACTO(copyright), from a solution that contains such proteins, in an active form. The factor VIII binding molecules of the present invention exhibit high affinity for factor VIII and factor VIII-like peptides. The current invention thus provides a cost-effective means for rapid purification of commercial quantities of proteins useful in the treatment of hemophilia A.
Accordingly, it is an object of the present invention to provide novel binding molecules for factor VIII and factor VIII-like proteins. Preferred binding molecules of the present invention exhibit not only distinct characteristics for binding of the target factor VIII polypeptides but also specific and desirable characteristics for release (elution) of the target polypeptides. Especially preferred binding molecules according to the invention are short polypeptide sequences, characterized by a stable loop structure.
A preferred method is disclosed herein for isolation of binding molecules according to the invention by employing phage display technology. The phage display method of the current invention is useful for identifying families of polypeptide binding molecules, and using this technique several binding peptides exhibiting high affinity for factor VIII and factor VIII-like peptides have been identified and isolated. Such binding peptides are useful for identifying, isolating and purifying factor VIII and factor VIII-like polypeptides from a solution.
The most preferred binding molecules specific for factor VIII and factor VIII-like peptides isolated by the phage display method of the present invention are polypeptides characterized by a loop structure formed as a result of a disulfide bond between two cysteine residues located at the positions disclosed in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Specific polypeptide binding molecules according to the present invention include polypeptides comprising amino acid sequences of the following general formulas:
X1-X2-Cys-X3-X4-X5-X6-X7-Cys-X8-X9 (SEQ ID NO: 1),xe2x80x83xe2x80x83I.
wherein X1 is Arg, Phe, His or Pro; X2 is Ser, Gly, Leu or His; X3 is Gly, Asn, Ile or Ser; X4 is Ser, Trp or Gly; X5 is Trp, Ile, Leu or Val; X6 is Phe, Trp or Ser; X7 is Pro or Phe; X8 is Ser, Leu, Pro or Phe; X9 is Ala, Phe, Leu or His;
X10-X11-Cys-X12-X13-Trp-X14-X15-Pro-Cys-X16-X17 (SEQ ID NO: 2),xe2x80x83xe2x80x83II.
wherein X10 is Arg or His; X11 is Ala, Arg, Gly, Leu or Pro; X12 is Gly or Phe; X13 is Ala or Ser; X14 is Leu or Phe; X15 is Arg, Asn or His; X16 is Ala, Asp, His, Leu, Phe, Pro, or Tyr; X17 is Ala, Arg, Asn, Asp, or His; and
Phe-Cys-X18-Val-X19-X20-Phe-X21-His-Cys-X22 (SEQ ID NO: 3),xe2x80x83xe2x80x83III.
wherein X18 is His or Trp; X19 is His or Phe; X20 is Ala, Asn, His, or Pro; X21 is Ala, Asn, Asp, Gln, His, Leu, Ser, or Val; X22 is Ala, Asp, His, Leu, Phe, or Ser.
In addition, it is also envisioned that the phage display method of the current invention can also be used to isolate additional families of binding molecules specific for factor VIII and factor VIII-like polypeptides.
The most preferred binding molecules for isolation and/or purification of factor VIII and factor VIII-like polypeptides, including especially REFACTO(copyright), mentioned above, from a solution include the following polypeptides:
His-Ser-Cys-Gly-Ser-Trp-Leu-Phe-Pro-Cys-Phe-Ala (SEQ ID NO: 4);
Phe-Gly-Cys-Ser-Trp-Leu-Phe-Pro-Cys-Pro-Phe (SEQ ID NO: 5);
Pro-His-Cys-Asn-Trp-Leu-Phe-Pro-Cys-Ser-Leu (SEQ ID NO: 6);
Arg-Leu-Cys-Ser-Trp-Ile-Ser-Pro-Cys-Ser-Ala (SEQ ID NO: 7);
Phe-His-Cys-Ile-Gly-Val-Trp-Phe-Cys-Leu-His (SEQ ID NO: 8);
Arg-Leu-Cys-Ser-Trp-Val-Ser-Pro-Cys-Ser-Ala (SEQ ID NO: 9);
His-Pro-Cys-Gly-Ser-Trp-Leu-Arg-Pro-Cys-Leu-His (SEQ ID NO: 10);
Arg-Gly-Cys-Gly-Ser-Trp-Leu-Arg-Pro-Cys-Leu-Asp (SEQ ID NO: 11);
His-Pro-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Ala-Ala (SEQ ID NO: 12);
His-Pro-Cys-Gly-Ser-Trp-Phe-Asn-Pro-Cys-Ala-His (SEQ ID NO: 13);
His-Pro-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-Phe-His (SEQ ID NO: 14);
His-Ala-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-His-Ala (SEQ ID NO: 15);
His-Leu-Cys-Gly-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala (SEQ ID NO: 16);
His-Leu-Cys-Phe-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala (SEQ ID NO: 17);
His-Gly-Cys-Gly-Ala-Trp-Phe-Arg-Pro-Cys-His-Ala (SEQ ID NO: 18);
His-Pro-Cys-Gly-Ala-Trp-Phe-Asn-Pro-Cys-Pro-Arg (SEQ ID NO: 19);
His-Pro-Cys-Gly-Ala-Trp-Leu-Arg-Pro-Cys-Tyr-Asn (SEQ ID NO: 20);
His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala (SEQ ID NO: 21);
Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp-His-Cys-His (SEQ ID NO: 22);
Phe-Cys-Trp-Val-His-Pro-Phe-Ala-His-Cys-Leu (SEQ ID NO: 23);
Phe-Cys-His-Val-Phe-His-Phe-Ser-His-Cys-Asp (SEQ ID NO: 24);
Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp-His-Cys-His (SEQ ID NO: 25);
Phe-Cys-Trp-Val-Phe-Asn-Phe-Ser-His-Cys-Ser (SEQ ID NO: 26);
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Asp (SEQ ID NO: 27);
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Ser (SEQ ID NO: 28);
Phe-Cys-Trp-Val-Phe-Pro-Phe-Gln-His-Cys-Ala (SEQ ID NO: 29);
Phe-Cys-Trp-Val-Phe-Pro-Phe-His-His-Cys-Phe (SEQ ID NO: 30);
Phe-Cys-His-Val-Phe-Asn-Phe-Val-His-Cys-Ser (SEQ ID NO: 31);
Phe-Cys-His-Val-Phe-Pro-Phe-Leu-His-Cys-Asp (SEQ ID NO: 32);
Solutions from which factor VIII and factor VIII-like polypeptides may be isolated and purified from include, but are not limited to, blood, blood fractions, and recombinant cell culture supernatants containing factor VIII or a factor VIII-like polypeptide produced and secreted by the recombinant host cell.
In another embodiment, the present invention provides a method for identifying and isolating factor VIII binding molecules via phage display technology. More specifically, the factor VIII and factor VIII-like binding molecules having specific and predetcrmined binding and elution characteristics may be selected from a binding molecule library, such as a phage display library, by a method comprising:
(a) selecting a first solution condition (i.e., the binding conditions) at which it is desired that a binding molecule should exhibit an affinity for factor VIII or a factor VIII-like polypeptide, forming an affinity complex;
(b) selecting a second solution condition (i.e., the release conditions) at which it is desired that the binding molecule will dissociate from the factor VIII or factor VIII-like polypeptide, wherein the second solution condition is different in some respect (e.g., temperature, pH, solvent concentration, etc.) from the first solution condition;
(c) providing a library of analogues of a parental factor VIII binding domain, wherein each analogue differs from said parental binding domain by variation of the amino acid sequence at one or more amino acid positions within the domain;
(d) contacting said library of analogues with factor VIII or a factor VIII-like polypeptide at the first solution condition under conditions suitable to form a complex between the binding molecule and a factor VIII or factor VIII-like polypeptide;
(e) removing from the solution the unbound members (analogues) of the binding domain library;
(f) subjecting the factor VIII or factor VIII-like polypeptide complexes that remain from step (e) to the second solution condition for dissociation of some of the binding molecule/factor VIII (or factor VIII-like polypeptide) complexes;
(g) recovering the binding analogues released under the second solution condition, wherein the recovered analogues identify isolated factor VIII or factor VIII-like binding molecules.
Optionally, the above procedure can include additional release condition steps, i.e., optionally subjecting the factor VIII or factor VIII-like polypeptide complexes that remain from step (f) to a third solution condition to dissociate other remaining complexes, which may be collected in a fraction separate from the factor VIII binding molecules released under the second solution conditions. Such a step, if the conditions are stringent enough to dissociate all of the complexes formed in step (d), will identify solution conditions suitable for regeneration of binding matrices utilizing the binding molecules isolated according to this process.
Also included in the present invention are non-peptide binding molecules and modified polypeptides that bind factor VIII and/or factor VIII-like polypeptides. An example of these modifications is a constrained-loop peptide having paired cysteine residues that form disulfide bonds, modified at the cysteine residues by substitution of one of the cysteines with non-natural amino acids capable of condensing with the other cysteine side-chain to form a stable thioether bridge. Such cyclic thioether analogues of synthetic peptides are described in PCT publication WO 97/46251, incorporated herein by reference. Other specifically contemplated modifications include specific amino acid substitutions to lend stability or other properties without significantly affecting factor VIII binding, e.g., substitution of Glu-Pro for Asp-Pro to reduce acid lability); N-terminal or C-terminal modifications to incorporate linkers such as poly-glycine segments and alterations to include functional groups, notably hydrazide (xe2x80x94NHxe2x80x94NH2) functionalities, e.g., to assist in immobilization of binding polypeptides according to this invention on solid supports.
In a further embodiment, the present invention encompasses a composition of matter comprising isolated nucleic acids, preferably DNA, encoding binding molecules of the present invention.
In another embodiment, the present invention provides a method for detecting a factor VIII or a factor VIII-like peptide in a solution suspected of containing it, comprising contacting the solution with a binding molecule according to the invention and determining whether a binding complex has formed.
A further embodiment of the present invention is a method for purification of factor VIII or a factor VIII-like polypeptide from a solution containing it, comprising the steps:
(a) contacting a solution containing factor VIII or a factor VIII-like polypeptide with a binding molecule according to this invention under solution conditions conducive to forming a binding complex comprised of factor VIII or a factor VIII-like polypeptide and the binding molecule;
(b) separating the complexes from the non-binding components of the solution;
(c) dissociating the factor VIII or factor VIII-like polypeptide from the binding molecule; and
(d) collecting the dissociated, purified factor VIII or factor VIII-like polypeptide.
Also envisioned by the present invention is a method for isolating factor VIII and factor VIII-like peptides comprising:
(a) immobilizing a binding molecule according to the invention on a solid support,
(b) contacting a factor VIII-containing solution or factor VIII-like polypeptide-containing solution with the solid support,
(c) removing the non-binding components from the solution, and
(d) eluting the factor VIII or factor VIII-like polypeptide from the solid support.
As used herein, the term xe2x80x9crecombinantxe2x80x9d is used to describe non-naturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated DNA and transformation of host cells. Recombinant is a term that specifically encompasses DNA molecules which have been constructed in vitro using genetic engineering techniques, and use of the term xe2x80x9crecombinantxe2x80x9d as an adjective to describe a molecule, construct, vector, cell, polypeptide or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides.
The term xe2x80x9cbacteriophagexe2x80x9d is defined as a bacterial virus containing a DNA core and a protective shell built up by the aggregation of a number of different protein molecules. The terms xe2x80x9cbacteriophagexe2x80x9d and xe2x80x9cphagexe2x80x9d are used herein interchangeably.
The term xe2x80x9cfactor VIII-like polypeptidexe2x80x9d is used to refer to a modified or truncated form of natural factor VIII or full-length recombinant factor VIII, which factor VIII-like polypeptide retains the procoagulant properties of factor VIII. Examples of factor VIII-like polypeptides are those active factor VIII fragments and factor VIII derivatives disclosed in the Andersson et al., Toole, and Almstedt et al. patents cited above, all of which are incorporated herein by reference. The term xe2x80x9cfactor VIII targetxe2x80x9d is sometimes used below to refer collectively to factor VIII and/or factor VIII-like polypeptides contained in a solution or production feed stream.
The term xe2x80x9cbinding moleculexe2x80x9d as used herein refers to any molecule, polypeptide, peptidomimetic or transformed cell (xe2x80x9ctransformantxe2x80x9d) capable of forming a binding complex with another molecule, polypeptide, peptidomimetic or tranformant. A xe2x80x9cfactor VIII binding moleculexe2x80x9d is a binding molecule that forms a complex with factor VIII. Specific examples of factor VIII binding molecules are the polypeptides described herein (e.g., SEQ ID NOs: 1-32) and bacteriophage displaying any of such polypeptides. Also included within the definition of factor VIII binding molecules are polypeptides derived from or including a polypeptide having an amino acid sequence according to formula I, II or III, above, and such polypeptides which have been modified for particular results. Specific examples of modifications contemplated are C-terminal or N-terminal amino acid substitutions or polypeptide chain elongations for the purpose of linking the binding moiety to a chromatographic support or other substrate, and substitutions of pairs of cysteine residues that normally form disulfide links, for example with non-naturally occurring amino acid residues having reactive side chains, for the purpose of forming a more stable bond between those amino acid positions than the former disulfide bond. All such modified binding molecules are also considered binding molecules according to this invention so long as they retain the ability to bind factor VIII and/or factor VIII-like polypeptides.
The present invention makes possible the highly selective detection or purification of factor VIII and/or factor VIII-like polypeptides in or from solutions containing them.
The factor VIII and factor VIII-like peptides may be produced in any known way, including chemical synthesis; production in transformed host cells; secretion into culture medium by naturally occurring cells or recombinantly transformed bacteria, yeasts, fungi, insect cells, and mammalian cells; secretion from genetically engineered organisms (e.g., transgenic mammals); or in biological fluids or tissues such as blood, plasma, etc. The solution that contains the crude factor VIII as it is initially produced (i.e., the production solution) will sometimes be referred to as the xe2x80x9cfeed streamxe2x80x9d.
Each method of producing factor VIII (or a factor VIII-like polypeptide) yields factor VIII in a feed stream that additionally contains a number of impurities (with respect to the factor VIII). One purpose of the present invention is to produce affinity ligands and preparations (such as chromatography media) comprising such ligands that allow rapid and highly specific purification of factor VIII from a particular feed stream. The factor VIII affinity ligands obtained herein may be tailored to the isolation of factor VIII from a particular feed stream, under specific preselected conditions. If an alternate production method for the factor VIIII is used, producing a different feed stream, a different set of affinity ligands may be necessary to achieve the same level of purification. The new set of ligands can be readily obtained following the procedures outlined herein.
Factor VIII binding molecules of the invention bind factor VIII with high affinity, comparable to or superior to other proteins such as antibodies known to bind factor VIII. Further, preferred affinity ligands described herein release the factor VIII intact and in active form under specific release conditions.
Selecting Binding and Release Conditions
Polypeptide binding molecules according to the present invention were isolated using phage display technology, in a manner to identify factor VIII binding peptides exhibiting particular preselected properties of binding and release. According to this methodology, two solution conditions may be preselected, i.e., binding conditions and release conditions. The binding conditions are a set of solution conditions under which it is desired that a discovered binding polypeptide will bind the target factor VIII (or factor VIII-like polypeptide); the release conditions are a set of solution conditions under which it is desired that a discovered binding polypeptide will not bind the factor VIII (i.e., will dissociate from factor VIII). The two conditions may be selected to satisfy any criterion of the practitioner, such as ease of attaining the conditions, compatibility with other purification steps, lowered cost of switching between conditions compared to other affinity media, etc. Preferably, the two solution conditions are selected so as not to adversely affect the stability or activity of the target protein (factor VIII or factor VIII-like polypeptide) and so as to differ significantly with respect to at least one solution parameter. For example, in conducting the screening for suitable binding peptides described herein, binders were selected that dissociated from the target in the presence of an ethylene glycol-containing buffer, or conditions of lowered pH (i.e., pH 2), or combinations of those conditions, which differed from the conditions employed for binding. Another parameter that could be advantageously varied is the concentration of a salt, for example NaCl, in the binding and elution buffers.
Selection of a Parental Binding Domain
In conjunction with selecting specific solution conditions for the desired binding and release of the factor VIII, a parental binding domain is selected to serve as a structural template for the engineered binding molecules that will exhibit the desired binding and release capabilities. The binding domain may be a naturally occurring or synthetic protein, or a region or domain of a protein. The parental binding domain may be selected based on knowledge of a known interaction between the parental binding domain and the factor VIII, but this is not critical. In fact, it is not essential that the parental binding domain have any affinity for factor VIII at all: Its purpose is to provide a structure from which a multiplicity of analogues (a xe2x80x9clibraryxe2x80x9d) can be generated, which multiplicity of analogues will include one or more analogues that exhibit the desired binding and release properties (and any other properties selected for). The binding conditions and the release conditions discussed supra may be selected with knowledge of the exact polypeptide that will serve as the parental binding domain, or with knowledge of a class of proteins or domains to which the parental binding domain belongs, or completely independently of the choice of the parental binding domain. Similarly, the binding and/or release conditions may be selected with regard to known interactions between a binding domain and the factor VIII, e.g., to favor the interaction under one or both of the solution conditions, or they may be selected without regard to such known interactions. Likewise, the parental binding domain can be selected taking into account the binding and/or release conditions or not, although it must be recognized that if the binding domain analogues are unstable under the binding or release conditions, no useful binding molecules may be obtained.
The nature of the parental binding domain greatly influences the properties of the derived peptides (analogues) that will be tested against the factor VIII molecule. In selecting the parental binding domain, the most important consideration is how the analogue domains will be presented to the factor VIII, i.e., in what conformation the factor VIII and the analogues will come into contact. In preferred embodiments, for example, the analogues will be generated by insertion of synthetic DNA encoding the analogue into a replicable genetic package, resulting in display of the domain on the surface of a microorganism, such as M13 phage, using techniques as described, e.g., in Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual, (Academic Press, Inc.; San Diego 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporated herein by reference.
For formation of phage display libraries, it is preferred to use structured polypeptides as the binding domain template, as opposed to unstructured, linear peptides. Mutation of surface residues in a protein will usually have little effect on the overall structure or general properties (such as size, stability, and temperature of denaturation) of the protein; while at the same time mutation of surface residues may profoundly affect the binding properties of the protein. The more tightly a peptide segment is constrained, the less likely it is to bind to any particular target. If it does bind, however, the binding is likely to be tighter and more specific. Thus, it is preferred to select a parental binding domain and, in turn, a structure for the polypeptide analogues, that is constrained within a framework having some degree of rigidity.
Preferably the protein domain that is used as the template or parental domain for generating the library of domain analogues will be a small protein or polypeptide. Small proteins or polypeptides offer several advantages over large proteins: First, the mass per binding site is reduced. Highly stable protein domains having low molecular weights, e.g., Kunitz domains (xcx9c7 kDa), Kazal domains (xcx9c7 kDa), Cucurbida maxima trypsin inhibitor (CMTI) domains (xcx9c3.5 kDa), and endothelin (xcx9c2 kDa), can show much higher binding per gram than do antibodies (150 kDa) or single-chain antibodies (30 kDa). Second, the possibility of non-specific binding is reduced because there is less surface available. Third, small proteins or polypeptides can be engineered to have unique tethering sites in a way that is impracticable for larger proteins or antibodies. For example, small proteins can be engineered to have lysines only at sites suitable for tethering (e.g., to a chromatography matrix), but this is not feasible for antibodies. Fourth, a constrained polypeptide structure is more likely to retain its functionality when transferred with the structural domain intact from one framework to another. For instance, the binding domain structure is likely to be transferable from the framework used for presentation in a library (e.g., displayed on a phage) to an isolated protein removed from the presentation framework or immobilized on a chromatographic substrate.
Immobilization of the polypeptides according to the invention is contemplated, e.g., onto chromatographic matrices to form efficient factor VIII separation media for solutions such as whole blood or conditioned culture media containing factor VIII secreted from a transformant host cell. By selecting appropriate binding domain templates, binding polypeptides having a single free (unpaired with another cysteine that ordinarily forms a disulfide link) cysteine can be isolated. Such thiol-functional polypeptides can be used for highly stable immobilization to substrates by formation of a thioether with iodoacetamide, iodoacetic acid, or similar xcex1-iodo carboxylic acid groups.
Similarly, the C-terminal carboxyl group of the polypeptide domain may be converted to a hydrazide (xe2x80x94NHxe2x80x94NH2), for reaction with an aldehyde-functional substrate or other amine-reactive substrate. This technique is preferred.
There are many small, stable protein domains suitable for use as parental binding domains and for which the following useful information is available: (1) amino acid sequence, (2) sequences of several homologous domains, (3) 3-dimensional structure, and/or (4) stability data over a range of pH, temperature., salinity, organic solvent, oxidant concentration. Some examples are: Kunitz domains (58 amino acids, 3 disulfide bonds), Cucurbida maxima trypsin inhibitor domains (31 amino acids, 3 disulfide bonds), domains related to guanylin (14 amino acids, 2 disulfide bonds), domains related to heat-stable enterotoxin IA from gram negative bacteria (18 amino acids, 3 disulfide bonds), EGF domains (50 amino acids, 3 disulfide bonds), kringle domains (60 amino acids, 3 disulfide bonds), fungal carbohydrate-binding domains (35 amino acids, 2 disulfide bonds), endothelin domains (18 amino acids, 2 disulfide bonds), and Streptococcal G IgG-binding domain (35 amino acids, no disulfide bonds). Most but not all of these contain disulfide bonds that maintain and stabilize the structure. The parental binding domain may also be based on a single loop (one disulfide) of a microprotein that is homologous to a known protein domain or not. For example, constrained loops of 7 to 9 amino acids were used to form libraries for isolating factor VIII and factor VIII-like polypeptide binding molecules, as described below. Libraries based on these domains, preferably displayed on phage, can be readily constructed and used for the selection of binding molecules according to this invention.
Providing a Library of Parental Binding Domain Analogues
Once a parental binding domain has been selected, a library of potential binding molecules is created for screening against a target, in this case factor VIII and/or factor VIII-like proteins, under the desired binding conditions and (optionally) the desired elution (release) conditions. The library is created by making a series of analogues, each analogue corresponding to the parental binding domain except having one or more amino acid substitutions in the amino acid sequence of the domain. The amino acid substitutions are expected to alter the binding properties of the domain without significantly altering its structure, at least for most substitutions. It is preferred that the amino acid positions that are selected for variation (variable amino acid positions) will be surface amino acid positions, that is, positions in the amino acid sequence of the domains which, when the domain is in its most stable conformation, appear on the outer surface of the domain (i.e., the surface exposed to solution). Most preferably the amino acid positions to be varied will be adjacent or close together, so as to maximize the effect of substitutions. In addition, extra amino acids can be added into the structure of the parental binding domain. In preferred embodiments, especially where a great deal of information is available concerning the interactions of factor VIII with other molecules, particularly the parental binding domain, those amino acid positions that are essential to binding interactions will be determined and conserved in the process of building the analogue library (i.e., the amino acids essential for binding will not be varied).
The object of creating the analogue library is to provide a very large number of potential binding molecules for reaction with the factor VIII molecule, and in general the greater the number of analogues in the library, the greater the likelihood that at least one member of the library will bind to the factor VIII and release under preselected or desirable conditions for release. Designed libraries following a particular template structure and limiting amino acid variegation at particular positions are much preferred, since a single library can encompass all the designed analogues and the included sequences will be known and presented in roughly equal numbers. By contrast, random substitution at only six positions in an amino acid sequence provides over 60 million analogues, which is a library size that begins to present practical limitations even when utilizing screening techniques as powerful as phage display. Libraries larger than this would pose problems in handling, e.g., fermentation vessels would need to be of extraordinary size, and more importantly, the likelihood of having all of the planned polypeptide sequence variations represented in the prepared library would decrease sharply. It is therefore preferred to create a designed or biased library, in which the amino acid positions designated for variation are considered so as to maximize the effect of substitution on the binding characteristics of the analogue, and the amino acid residues allowed or planned for use in substitutions are limited, e.g., on the basis that they are likely to cause the analogue to bind under the solution conditions at which the library will be screened for binders.
As indicated previously, the techniques discussed in Kay et al., supra, and Ladner et al. U.S. Pat. No. 5,223,409 are particularly useful in preparing a library of analogues corresponding to a selected parental binding domain, which analogues will be presented in a form suitable for large-scale screening of large numbers of analogues with respect to a target factor VIII molecule. The use of replicable genetic packages, and most preferably bacteriophage, is a powerful method of generating novel polypeptide binding entities that involves introducing a novel, exogenous DNA segment into the genome of a bacteriophage (or other amplifiable genetic package) so that the polypeptide encoded by the non-native DNA appears on the surface of the phage. When the inserted DNA contains sequence diversity, then each recipient phage displays one variant of the template (parental) amino acid sequence encoded by the DNA, and the phage population (library) displays a vast number of different but related amino acid sequences.
In a screening procedure to obtain factor VIII binders according to this invention, a phage library is contacted with and allowed to bind a target factor VIII molecule, usually immobilized on a solid support. Non-binders are separated from binders. In various ways, the bound phage are liberated from the factor VIII, collected and amplified. Since the phage can be amplified through infection of bacterial cells, even a few binding phage are sufficient to reveal the gene sequence that encodes a binding entity. Using these techniques it is possible to recover a binding phage that is about 1 in 20 million in the population. One or more libraries, displaying 10-20 million or more potential binding polypeptides each, can be rapidly screened to find high-affinity factor VIII binders. When the selection process works, the diversity of the population falls with each round until only good binders remain, i.e., the process converges. Typically, a phage display library will contain several closely related binders (10 to 50 binders out of 10 million). Indications of convergence include increased binding (measured by phage titers) and recovery of closely related sequences. After a first set of binding peptides is identified, the sequence information can be used to design other libraries biased for members having additional desired properties, e.g., discrimination between factor VIII and particular fragments or closely related impurities in a particular feed stream.
Such techniques make it possible not only to screen a large number of potential binding molecules but make it practical to repeat the binding/elution cycles and to build secondary, biased libraries for screening analog-displaying packages that meet initial criteria. Using these techniques, an analogue biased library may be screened to reveal members that bind tightly (i.e., with high affinity) under the screening conditions.
Synthesis of Polypeptide Analogues
Following the procedures outlined above, additional binding molecules for factor VIII and/or factor VIII-like polypeptides may be isolated from the phage display libraries described herein or other phage display libraries or collections of potential binding molecules (e.g., combinatorial libraries of organic compounds, random peptide libraries, etc.). Once isolated, the sequence of any individual binding peptide or the structure of any binding molecule can be analyzed, and the binder may be produced in any desired quantity using known methods. For example, the polypeptide binding molecules described herein, since their sequences are now known, may advantageously be produced by chemical synthesis followed by treatment under oxidizing conditions appropriate to obtain the native conformation, i.e., the correct disulfide bond linkages. Synthesis may be carried out by methodologies well known to those skilled in the art (see, Kelley et al. in Genetic Engineering Principles and Methods, (Setlow, J. K., ed.), Plenum Press, NY., (1990) vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989), W. H. Freeman Co., San Francisco). The binding molecules of the present invention can be made either by chemical synthesis or by semisynthesis. The chemical synthesis or semisynthesis methods allow the possibility of non-natural amino acid residues to be incorporated.
Polypeptide binding molecules of the present invention are preferably prepared using solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85: 2149 (1963); Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132 (1985)). Solid phase synthesis begins at the carboxy-terminus of the putative polypeptide by coupling a protected amino acid to a suitable resin, which reacts with the carboxy group of the C-terminal amino acid to form a bond that is readily cleaved later, such as a halomethyl resin, e.g., chloromethyl resin and bromomethyl resin, hydroxymethyl resin, aminomethyl resin, benzhydrylamine resin, or t-alkyloxycarbonyl-hydrazide resin. After removal of the xcex1-amino protecting group with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, TEA, the next cycle in the synthesis is ready to proceed. The remaining (xcex1-amino and, if necessary, side-chain-protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the resin. Alternatively, some amino acids may be coupled to one another forming an oligopeptide prior to addition of the oligopeptide to the growing solid phase polypeptide chain.
The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as azide method, mixed acid anhydride method, DCC (dicyclohexylcarbodiimide) method, active ester method (p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino)phosphonium hexafluorophosphate]method, N-hydroxysuccinic acid imido ester method), and Woodward reagent K method.
Common to chemical synthesis of peptides is the protection of the reactive side-chain groups of the various amino acid moieties with suitable protecting groups at that site until the group is ultimately removed after the chain has been completely assembled. Also common is the protection of the xcex1-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group followed by the selective removal of the xcex1-amino-protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in the desired sequence in the polypeptide chain with various of these residues having side-chain protecting groups. These protecting groups are then commonly removed substantially at the same time so as to produce the desired resultant product following purification.
The typical protective groups for protecting the xcex1- and xcex5-amino side chain groups are exemplified by benzyloxycarbonyl (Z), isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(NO2)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl, adamatyloxycarbonyl, 2-(4-biphenyl)-2-proploxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyiethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl (Mpt), and the like.
As protective groups for the carboxy group there can be exemplified, for example, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group as occasion demands. For example, the guanidino group in arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds), 1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group in cysteine may be protected with p-methoxybenzyl, triphenylmethyl, acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethy-benzyl (Tmb), etc., and the hydroxyl group in the serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl, etc.
After the desired amino acid sequence has been completed, the intermediate polypeptide is removed from the resin support by treatment with a reagent, such as liquid HF and one or more thio-containing scavengers, which not only cleaves the polypeptide from the resin, but also cleaves all the remaining side-chain protecting groups. Following HF cleavage, the protein sequence is washed with ether, transferred to a large volume of dilute acetic acid, and stirred at pH adjusted to about 8.0 with ammonium hydroxide. Upon pH adjustment, the polypeptide takes its desired conformational arrangement.
Polypeptides according to the invention may also be prepared commercially by companies providing polypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals, Inc., Hopkinton, Mass.).
Use of the Binding Molecules in Detection or Purification
For detection of factor VIII and/or factor VIII-like polypeptides in a solution such as blood or conditioned media suspected of containing it, a binding molecule according to the invention can be detectably labeled, e.g., radiolabeled or enzymatically labeled, then contacted with the solution, and thereafter formation of a complex between the binding molecule and the factor VIII target can be detected. A phage binding molecule according to the invention, i.e., a recombinant phage displaying a factor VIII binder polypeptide on its surface, may form a complex with factor VIII and/or factor VIII-like polypeptides that is detectable as a sediment in a reaction tube, which can be detected visually after settling or centrifugation.
Alternatively, a sandwich-type assay may be used, wherein a factor VIII binding molecule is immobilized on a solid support such as a plastic tube or well, or a chromatographic matrix such as sepharose beads, then the solution suspected of containing the factor VIII target is contacted with the immobilized binding molecule, non-binding materials are washed away, and complexed factor VIII or factor VIII-like polypeptide is detected using a suitable detection reagent, such as a monoclonal antibody recognizing the factor VIII target, which reagent is detectable by some conventional means known in the art, including being detectably labeled, e.g., radiolabeled or labeled enzymatically, as with horseradish peroxidase, and the like.
The binding molecules according to this invention will be extremely useful for isolation of factor VIII and/or factor VIII-like polypeptides by affinity chromatography methods. Any conventional method of chromatography may be employed. Preferably, an affinity ligand of the invention will be immobilized on a solid support suitable, e.g., for packing a chromatography column. The immobilized affinity ligand can then be loaded or contacted with a feed stream under conditions favorable to formation of binding molecule/factor VIII (or factor VIII-like polypeptide) complexes. Non-binding materials can be washed away, then the factor VIII (or factor VIII-like polypeptide) can be eluted by introducing solution conditions favoring dissociation of the binding complex.
Alternatively, batch chromatography can be carried out by mixing a solution containing the factor VIII target and the binding molecule, then isolating complexes of the factor VIII target and the binding molecules. For this type of separation, many methods are known. For example, the binding molecule can be immobilized on a solid support, then separated from the feed stream along with the factor VIII target by filtration. Or the binding molecule may be modified with its own affinity tag, such as a polyHis tail, which can be used to bind the binder after complexes have formed using an immobilized metal affinity chromatography. Once separated, the factor VIII target can be released from the binding molecule under elution conditions and recovered in pure form.
It should be noted that although precise binding conditions were preselected in obtaining the factor VIII-binding polypeptides disclosed herein, subsequent use in affinity purification may reveal more optimal binding and release conditions under which the same isolated affinity ligand will operate. Thus, it is not critical that the binding molecule, after isolation according to this invention, be always employed only at the binding and release conditions that led to its separation from the library.

Binding molecules for human factor VIII and factor VIII-like proteins


A semiconductor memory device includes a memory which comprises a confidential information area storing confidential information and a flag. A controller reads the flag from the memory when instructed to erase or write data in the confidential information area, determines whether the flag is set, erases or writes data in the confidential information area when the flag is clear, and abandons a process requested by an erase or write instruction when the flag is set. An authenticator uses data in the confidential information area to execute an operation for authentication. A management information area may store management information for associated pages. The flag may include a bit string and a complementary bit string to improve reliability of the flag. The confidential information area may store dummy data when the memory is used for uses other than an application with an authentication function, so no problem arises using a normal controller.

Storing content, such as video, in media which incorporates a NAND flash memory has been widely practiced in recent years. In order to protect the content rights, functionalities to authenticate such media by host devices, such as video players, have been developed. For example, the host devices determine whether the media is an authorized or unauthorized copy and refuses to play the video if the media is the latter. Data required for authentication, such as a secret key, is written to a specific area in the NAND flash memory before shipment. If such data in the specific area can be rewritten by any user, an attacker can erase the area and write data which can pass the authentication to duplicate unauthorized media.

Semiconductor memory device including a flag for selectively controlling erasing and writing of confidential information area


A power system for a machine is disclosed. The power system may have an engine, and an operator input device controllable to affect operation of a plurality of different tools that can be removably attached to the machine. The plurality of different tools may include at least a first tool capable of consuming about 50% or more of a total power output of the engine. The power system may also have a controller configured to detect attachment of the at least a first tool to the machine and, based on a signal from the operator input device, selectively increase a power output of the engine when the at least a first tool is attached to the machine. The controller may also be configured to selectively decrease the power output of the engine when the at least a first tool is not attached to the machine.

Mobile construction and excavation machines, such as skid-steer loaders, integrated tool carriers, excavators, and other machines, are often capable of supporting and powering a variety of different tools to perform any number of assigned tasks. Some of these tools require extra electric or fluid power to operate. Onboard generators or pumps driven by a machine's engine provide the extra power to the tools during this time of need. Powered tools can be divided into at least two categories, including low-power tools that require a relatively low level of auxiliary power to operate effectively, and high-power tools that require higher amounts of auxiliary power. Accordingly, the engine of a machine capable of attachment to high-power tools should also be capable of relatively high power output.
Problems can occur when high-power machines that are capable of operation with high-power tools are operated with low-power tools or without any tools at all. For example, a skid-steer loader having an engine capable of producing high-levels of power, when operated with low-power tools or without tools, can direct too much power to traction devices of the machine. This excessive power directed to the traction devices can cause the traction devices to spin, thereby making the machine difficult to control and/or causing premature wear of machine components and lower machine efficiency.
An exemplary machine capable of operation with different types of tools is described in U.S. Pat. No. 6,397,571 of Ehrecke that issued on Jun. 4, 2002 ("the '571 patent"). Specifically, the '571 patent discloses a combine having an electronic engine controller that stores two power curves for controlling an engine. The first power curve has a higher horsepower output than the second power curve. A harvesting assembly detector signals the electronic engine controller with a corn head signal when a corn head is mounted to the combine, or a grain head signal when a grain platform is mounted to the combine. When a corn head is mounted to the combine, the electronic engine controller derates the engine and works on the second power curve. When the electronic engine controller receives a grain platform signal, the electronic engine controller works on the higher horsepower first power curve. Detection of the different harvesting assemblies can be accomplished by electronic hookup between each harvesting assembly and the combine.
Although the combine of the '571 patent may be capable of adjusting engine operation based on tool configuration, it may be less than optimal. In particular, the '571 patent does not disclose any action being taken when no tools are connected to the combine or even if operation without a tool is possible. Further, the '571 patent may not consider operator input when adjusting engine operation, which may cause undesired performance in some situations. Finally, detection of the different harvesting assemblies may be complex and/or prone to error.
The power system of the present disclosure is directed toward solving one or more of the problems set forth above and/or other problems of the prior art.

Power system having attachment-based engine control


The disclosed computer-implemented method may include receiving, by a transportation requestor device, progress information that indicates progress of a transportation provider toward a request location, presenting, via a graphical user interface of the transportation requestor device, the progress of the transportation provider toward the request location with a graphical representation of the transportation provider according to the progress information, receiving, by the transportation requestor device, contextual information from a server that explains the progress of the transportation provider as indicated by the progress information, and changing, via the graphical user interface of the transportation requestor device, the graphical representation of the transportation provider with a graphical representation of the contextual information. Various other methods, systems, and computer-readable media are disclosed.

Some transportation services may provide transportation to transportation requestors using transportation providers to meet the needs of those requesting transportation services on an on-demand basis. However, while on-demand transportation services may provide convenience and flexibility to those requesting transportation, some on-demand transportation services may also create confusion, uncertainty, or anxiety while transportation requestors wait for the transportation to arrive due to the lack of sufficient transportation provider travel progress information being provided to transportation requestor devices.
Throughout the drawings, identical reference characters and descriptions indicate similar, but not necessarily identical, elements. While the exemplary embodiments described herein are susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. However, the exemplary embodiments described herein are not intended to be limited to the particular forms disclosed. Rather, the instant disclosure covers all modifications, equivalents, and alternatives falling within the scope of the appended claims.

Apparatuses, systems, and methods for graphical progress interfaces for dynamic transportation networks


A lithium air battery module including a lithium air battery cell including a first electrolyte; an additional electrolyte disposed non-adjacent the first electrolyte; and a housing which accommodates the lithium air battery cell and the additional electrolyte.

1. Field
The present disclosure relates to a lithium air battery module.
2. Description of the Related Art
Lithium air batteries include a negative electrode capable of reversibly discharging lithium ions, a positive electrode that includes an oxidation-reduction catalyst and use oxygen in air as a positive electrode active material, and a lithium ion conductive medium disposed between the positive electrode and the negative electrode.
A specific energy of a lithium air battery is greater than 3000 watt hours per kilogram (Wh/kg), which corresponds to a specific energy about 10 times higher than that of a lithium ion battery. In addition, a lithium air battery is eco-friendly and may provide improved stability compared to a lithium ion battery, and thus development of a lithium air battery is being actively conducted.
Lithium metal has high capacity, and thus is a desirable negative electrode active material for a lithium air battery.
However, since a lithium air battery uses an oxygen-containing gas, e.g., air, as the positive electrode active material, a positive electrode of the lithium air battery is open to the environment. Because the lithium air battery in open to the environment, an electrolyte contacting the positive electrode can evaporate, reducing a lifespan of the lithium air battery due to evaporation of the electrolyte. Thus there remains a need for a lithium air battery having improved lifespan.


A slip on attachment for use on footwear when additional traction is needed has a sole, a heel seat and resilient arms. The sole is divided by a slit opening through the toe portion and extending into the heel seat and is resiliently yieldable so that it will spread apart as footwear is inserted between and under the arms until the heel of the footwear can enter the heel seat, the arms then engaging and holding the footwear in its waist line zone. The sections of the sole are provided with anti-slip portions.

While there are other surfaces than icy ones where better traction is desirable or needed for those traveling on foot over them, the present invention is disclosed herein with particular reference to icy conditions.
For many years, so called "ice creepers" have been available to enable ice to be walked on more safely. These, as far as I am aware have been and still are held on footwear by straps which, when buckled together, hold the ice creepers on the footwear. Such attachments, while generally satisfactory in use, require approximately the same procedures as is required to don conventional footwear. Such attachments, however, often are inconvenient to attach.
Footwear is, of course, available which is to be worn and provide improved traction on various surfaces. Shoes for golfers, track and field uses, and baseball and football players are examples which illustrate the different types of protuberances which are employed to provide adequate traction on different surfaces.

Split-sole anti-slip attachments for footwear


A culvert collar for providing a device that will support a culvert pipe and further reduces the installation time and maintenance time when working with culvert pipes. The inventive device includes a two piece flange assembly that has a first member that has a plurality of tongue portions and a second member with tongue grooves. The first member is placed in position in the trench and the culvert pipe is seated in a semi-circular channel of the first member. The second member is placed over the culvert pipe and held in position by the coupling of the tongues portions with the tongue grooves.

1. Field of the Invention
The present invention relates to culvert pipe supports and more particularly pertains to a new culvert collar for providing a device that the will support a culvert pipe and further reduces the installation time and maintenance time when working with culvert pipes.
2. Description of the Prior Art
The use of culvert pipe supports is known in the prior art. More specifically, culvert pipe supports heretofore devised and utilized are known to consist basically of familiar, expected and obvious structural configurations, notwithstanding the myriad of designs encompassed by the crowded prior art which have been developed for the fulfillment of countless objectives and requirements.
Known prior art culvert pipe supports include U.S. Pat. No. 5,252,002; U.S. Pat. No. 4,983,070; U.S. Pat. No. 4,993,872; U.S. Pat. No. 4,930,937; and U.S. Pat. No. 5,123,777.
While these devices fulfill their respective, particular objectives and requirements, the aforementioned patents do not disclose a new culvert collar. The inventive device includes a first and second member that is releasable interlocked around the culvert pipe for support and protection.
In these respects, the culvert collar according to the present invention substantially departs from the conventional concepts and designs of the prior art, and in so doing provides an apparatus primarily developed for the purpose of providing a device that will support a culvert pipe and further reduces the installation time and maintenance time when working with culvert pipes.


The invention provides a method for driving a graphic processing unit (GPU), where a driver applies two threads to drive one ore more GPUs. The method includes the steps of: (a) activating a rendering thread and a displaying thread in response to invoking by an application thread of a graphics application; (b) sending according to the rendering thread a plurality of rendering instructions for enabling generation of at least a first rendered frame and a second rendered frame; and (c) sending according to the displaying thread one or more interpolating instructions and one or more displaying instructions, the one or more interpolating instructions enabling execution of interpolation according to the at least a first rendered frame and the second rendered frame to create one or more interpolated frames, and the one or more displaying instructions enabling display of the one or more interpolated frames.

1. Field of the Invention
The present invention relates to a method for driving a graphics processing unit (GPU).
2. Description of the Related Art
Conventionally a graphics processing unit (GPU) has to finish processing the current frame before it starts processing the next frame according to an instruction from a graphics driver. In general, frame processing comprises rendering (or similar calculation, for example interpolation) and displaying (that is, sending a rendered or calculated frame to a frame buffer in which the rendered or calculated frame waits for the display thereof).
Therefore, it is only after the current frame has been sent to a frame buffer that the GPU is able to process an instruction given by the graphics driver and related to the next frame.

Method for graphics driver level decoupled rendering and display


A fluid coupling reliably finding absence of the gasket, if exists, in the leak test is provided. Butted end surfaces of coupling members have annular sealing protrusions and anti-overfastening annular protrusions protruded further than the annular sealing protrusions, respectively. At least one of the anti-overfastening annular protrusions has a communication path communicating internal space with external space when both the anti-overfastening annular protrusions butt against each other.

A conventional fluid coupling includes first and second coupling members having respective fluid paths communicating with each other, an annular gasket interposed between butted end surfaces of both the coupling members, and a screw means that couples the coupling members to each other, and the butted end surface of each coupling member has an annular sealing protrusion and an anti-overfastening annular protrusion protruded further than the sealing protrusion (for example, Patent Document 1).
In the fluid coupling, the annular sealing protrusions come into contact with the gasket to deform the gasket, thereby obtaining required sealing property, and the anti-overfastening annular protrusions prevent fastening exceeding proper fastening. At proper fastening, the anti-overfastening annular protrusion of one coupling member is opposed to the anti-overfastening annular protrusion of the other coupling member via a retainer or not via the retainer, and the anti-overfastening annular protrusions do not exert any force to each other or do not exert a substantial force to each other. To prevent overfastening, at further fastening, the anti-overfastening annular protrusion of one coupling member and the anti-overfastening annular protrusion of the other coupling member butt against each other via the retainer or directly, increasing a resistance to fastening, thereby preventing excessive fastening.


The invention provides methods and compositions for treating subjects using Grb7 antagonists. Specifically disclosed are Grb7 antagonists that bind selectively to Grb7 and interfere with the ability of Grb7 to bind to its native ligands. These compositions are useful in the prevention and treatment of disorders characterized by abnormal interaction of Grb7 with its native ligands (e.g., ErbB2).

Signal transduction is the process by which extracellular signals are transduced into a cell, and thereby impact upon process such as cell proliferation and differentiation. Tyrosine phosphorylation is a common mechanism for transducing a signal between signaling factors. Tyrosine kinases are enzymes which are capable of phosphorylating peptides and polypeptides at tyrosine residues. A subset of tyrosine kinases is also capable of dimerizing and thereby phosphorylating itself. Phosphatases are enzymes, which can optionally be receptors as well, capable of dephosphorylating a molecule such as a signaling factor. Uncontrolled tyrosine kinase activation has been implicated in the increased proliferation of cancerous cells.
In addition to tyrosine kinases and phosphatases, signal transduction pathways also use adaptor proteins. Adaptor proteins are intracellular proteins that act as intermediary proteins. These proteins allow for the association of other, usually signaling, proteins which would not otherwise be capable of interacting with each other. Adaptor proteins commonly do not have an activity other than their "adaptor" function, although they may act as substrates for other proteins such as, for example, kinases. Many adaptor proteins share conserved protein interaction domains such as, for example, Src homology (SH) domains. SH2 domains recognize and bind to phosphorylated tyrosine residues.
Abnormal signal transduction has been associated with a variety of disorders. A number of oncogenes are mutant variants of normally occurring signaling factors, including tyrosine kinases. One strategy for preventing or treating such disorders is to interfere with the unregulated or increased signal transduction which is thought to occur in these disorders.

Methods and compositions for inhibiting GRB7

University of Vermont and State Agricultural College


An audio-signal-processing apparatus includes a band-decomposition unit utilizing a band-decomposition characteristic based on the harmonic-tone composition of musical instruments. A low interval limit generates the harmonic-tone to each band signal after decomposing the bass signal into a plurality of band signals. The audio-signal processing apparatus generates a harmonic-tone individually for each pure tone component, which includes complex tones and suppresses deterioration of the sound quality.

1. Field of the Invention
This invention relates to an audio-signal-processing apparatus capable of harmonic-series-generation and a method thereof.
2. Description of the Related Art
Audio-signal-processing apparatuses with harmonic-series-generation have been proposed for various purposes. Generally known is, for example, an effector apparatus that gives variations to tone color of a musical instrument and voice, especially, a virtual reproduction system of bass sound that generates a harmonic-tone for a bass component to reinforce the bass sounds in a small speaker.
A prior art virtual reproduction system of bass sound will be described with reference to FIG. 9 through FIG. 13. FIGS. 9 (a) and (b) are block diagrams illustrating a conventional audio-signal-processing apparatus. Each harmonic-tone can be generated with a full-wave-rectification method, a power method, a zerocrossing method or other similar method.
A first example of a prior art system will be described with reference to FIG. 9 (a). In this example, a plurality of harmonic-tone components of integer orders are generated with the zerocrossing method or the power method for low frequency components.
As illustrated in FIG. 9 (a), a signal inputted from an input terminal 91 is divided into two systems. In the first system, all the components of the input signal are inputted into a delay unit 93, and the output of the delay unit is then inputted into one input of an adder 100 after undergoing gain adjustment by a gain-control 94a. The components of the input signal are delayed by a time that is required for processing in a harmonic-series-generating unit 97, whose processing will be described later.
In the second system, all the components of the input signal are inputted into a low pass filter 96. The low pass filter 96 extracts only low frequency components from all these components according to a predetermined cut-off characteristic of the low pass filter 96, and outputs the extracted low frequency components to harmonic-series-generating unit 97.
Throughout this specification, a harmonic-tone with n times the frequency (n is a natural number) of the fundamental tone frequency (fundamental frequency) is referred to as an n-th harmonic-tone.
The harmonic-series-generating unit 97 includes harmonic-series-generating units 98a, 98b, . . . , 98c that generate a 2nd harmonic-tone, a 3rd harmonic-tone, . . . , an n-th harmonic-tone for the low frequency components that the low pass filter 96 has extracted. The 2nd harmonic-tone and the 3rd harmonic-tone, . . . , the n-th harmonic-tone that these harmonic-series-generating units 98a, 98b, . . . , 98c generate are inputted into another input of the adder 100 after undergoing gain adjustment by gain controls 94b, 94c, . . . , 94d respectively and then added in an adder 95.
The adder 100 adds the components inputted from the first system and the second system respectively and outputs the sum to an output terminal 92.
Another example of a prior art system will be described with reference to FIG. 9 (b). In this example, a harmonic-tone is generated for the low frequency component with the full-wave-rectification method. Japanese Patent Publication No. 05-328481 discloses a circuit pertaining to the second example.
In FIG. 9 (b), the explanation is omitted for the same components described in and with reference to FIG. 9 (a).
In FIG. 9 (b), the harmonic-series-generating unit 97 illustrated in the FIG. 9 (a) is replaced with a full wave rectifier 99 and a band pass filter 101.
The full wave rectifier 99 changes negative values of the low frequency signals that a low pass filter 96 has extracted to positive values, doubling the frequency of the low frequency signals. However, since direct-current bias and even harmonic-tone components are actually generated, only the 2nd harmonic-tone that is a principal component, is extracted using the band pass filter 101.
As illustrated in FIG. 9 (b), full-wave-rectification can be executed with a simple structure. The full-wave-rectification system can generate a second harmonic-tone and be expanded to generate harmonic-series on the order of an n-th power of 2 (n=2, 3, . . . ) by cascading plural units of the full-wave-rectification 99 and using a band pass filter 101 as illustrated in FIG. 9 (c). However, this full-wave-rectification system poses a problem in that odd harmonic-tones cannot be generated.
Next, problems associated with the zerocrossing method and the first example in the prior art will be described with reference to FIG. 10. FIGS. 10 (a) and (b) illustrate graphs exemplifying waveforms resulting from the prior art zerocrossing method the prior art. In the figures, the horizontal axis illustrates time and the vertical axis indicates amplitude.
The zerocrossing method detects the zerocrossing points P1, P2, and P3 as the signal changes from positive to negative or from negative to positive. These detected zerocrossing points are illustrated by the circles depicted in FIG. 10 (a).
In generating the second harmonic-tone component, a signal is double-compressed in the time axis and repeatedly reproduced twice in an interval between one zerocrossing point and the next zerocrossing point (for example, an interval between P1 and P2 or an interval between P2 and P3, in FIG. 10 (a)).
Similarly, an n-th order harmonic-tone is obtained by compressing a signal n times and repeatedly reproducing the signal n times in an interval between two consecutive zerocrossing points on the time axis.
A general music source is a complex tone that includes a plurality of pure tone components. For example, a periodical musical sound is includes a fundamental tone with (f=40 Hz in FIG. 11 (a)) and harmonic-tones with frequencies of integral multiples of the fundamental frequency (80, 120 . . . Hz), as illustrated in spectrum structure examples in FIG. 11 (a).
Further, a chord possesses a plurality of fundamental frequencies with strong energy components. For example, the chord possesses the spectrum structure illustrated in FIG. 11 (b) in Perfect 8th, and the spectrum structure illustrated in FIG. 11 (c) in Perfect 5th.
Next, problems encountered when using conventional harmonic-series-generating methods for such general complex tones (including a plurality of pure tones) will be described. Hereafter, a zerocrossing method used as a harmonic-series-generating method will be described. Additionally, problems encountered when using other harmonic-series-generating methods, such as the full-wave-rectification method and the power methods, will be described.
FIG. 12 (a) illustrates the results of the frequency analysis performed on the processed sounds, after generating the 2nd harmonic-tone at the same level as the original signal, by the zerocrossing method for complex tones (refer to FIG. 11 (b) regarding its spectrum structure). The complex tones include two pure tones (pure tones of 40 Hz and 80 Hz of the same level) that are in the Perfect 8th relation (the frequency ratio is 1:2).
FIG. 12 (b) illustrates the results of the frequency analysis performed on the processed sounds, after generating the 2nd harmonic-tone at the same level as the original signal, by the zerocrossing method for complex tones (refer to FIG. 11 (c) regarding its spectrum structure). The complex tones include two pure tones (pure tone of 60 Hz and 90 Hz of the same level) that are in the Perfect 5th relation (the frequency ratio is 2:3).
It is desirable for the processed sounds to possess only the second harmonic components of each pure tone component in both FIGS. 12 (a) and (b). However, although the second harmonic components are generated in either case, some distortions occurs due to the inclusion of other components than the second harmonic components (e.g., the fundamental frequency equal to the highest common factor of the fundamental frequency of two pure tone components). This distortion occurs since the harmonic-tone is generated at once for the signal with a plurality of pure tones.
As illustrated in FIG. 13 (a), in a signal with a plurality of pure tones, the waveform cycle T of the complex tone (the least common multiple of the contained pure tone cycles) and the interval of the zerocrossing points in the original waveform do not usually agree with each other.
For such a case, applying the zerocrossing method compresses the waveform and makes reproduction repeatedly in other interval than the zerocrossing points with cycle T, as illustrated in FIG. 13 (c). As a result, the processed signal waveform is not similar to the original signal and waveform distortion occurs. Comparing FIG. 13 (b) with FIG. 13 (c), FIG. 13 (b) illustrates the waveform that has the ideal harmonic-series-generation of the original waveform of FIG. 13 (a). Therefore, there is a need to overcome or lessen the disadvantages noted above in the prior art.

Apparatus and method for generating harmonics in an audio signal

Matsushita Electric Industrial Co., Ltd.


The recognition of text in an acquired image is improved by using general and type-specific heuristics that can determine the likelihood that a portion of the text is truncated at an edge of an image, frame, or screen. Truncated text can be filtered such that the user is not provided with an option to perform an undesirable task, such as to dial an incorrect number or connect to an incorrect Web address, based on recognizing an incomplete text string. The general and type-specific heuristics can be combined to improve confidence, and the image data can be pre-processed on the device before processing with an optical character recognition (OCR) engine. Multiple frames can be analyzed to attempt to recognize words or characters that might have been truncated in one or more of the frames.

Recent years have seen drastic increases in the use of portable computing devices, such as smart phones and tablet computers. Today's consumers are utilizing such devices for a wide variety of purposes, such as to research items on the Internet, purchase products and services, capture and/or send digital images, compose electronic mail (email) messages, make telephone calls, and the like. One particular area of attention includes the ability to process image data captured by one or more digital cameras often embedded in such devices, in order to perform various actions based on the information in the image. For example, if the image contains an object that can be recognized as a product, the computing device may invoke an application to purchase the product from an electronic commerce (e-commerce) provider. Similarly, if the image contains an object recognized as a place of business (e.g., restaurant, bar, etc.) or an address, the computing device may invoke a map application to display directions to the user. Many other examples of such image processing are also possible.
In this image processing context, problems may arise when the camera of the computing device is aimed in such a way as to capture only a portion of the object, where the remaining portion of the object is outside of the field of view of the camera. In particular, cutting off portions of certain text in such a manner may cause the computing device to perform actions that are erroneous or do not conform to the expectations of the user.


In general, in a first aspect, the invention features a system that includes a microlithography projection optical system. The microlithography projection optical system includes a plurality of elements arranged so that during operation the plurality of elements image radiation at a wavelength λ from an object plane to an image plane. At least one of the elements is a reflective element that has a rotationally-asymmetric surface positioned in a path of the radiation. The rotationally-asymmetric surface deviates from a rotationally-symmetric reference surface by a distance of about λ or more at one or more locations of the rotationally-asymmetric surface.

Projection objectives are widely used in microlithography to transfer a pattern from a reticle to a substrate by forming an image of the reticle on a layer of a photosensitive material disposed on the substrate. In general, projection objectives fall into three different classes: dioptric objectives; catoptric objectives; and catadioptric objectives. Dioptric objectives use refractive elements (e.g., lens elements) to image light from an object plane to an image plane. Catoptric objectives use reflective elements (e.g., mirror elements) to image light from an object plane to an image plane. Catadioptric objectives use both refractive and reflective elements to image light from an object plane to an image plane.

Catoptric objectives and systems using catoptric objectives


A welded assembly includes a weld joint formed via a spin-welding process. A disc receives a cylinder prior to spin-welding, with the disc having a circumferential groove undercutting or defining an annular shelf. An interlocking feature retaining the cylinder and disc is formed between a flow pattern of the cylinder and the annular shelf upon cooling of molten flash in the groove underneath the annular shelf. Teeth can be formed integrally with the disc to provide a torsional interlocking feature between the disc and cylinder. A method for forming a weld joint between a plastic cylinder and disc includes providing the disc with a circumferential groove forming an annular shelf, and rotating the cylinder with respect to the disc under an axial force to thereby form an outflow of molten flash. The flash forms an interlocking feature when cooled after flowing into the groove underneath the shelf.

In a friction-welding process, such as conventional spin-welding process, the components to be welded or fused are placed adjacently to each other and then rotated at a high relative rate of speed in conjunction with an applied axial clamping force. The frictional heating generated at or along a boundary or interface between the components melts a portion of the material, typically plastic or metal, which then flows away from the interface or the "weld zone" in molten form, hereinafter referred to as "molten flash". When the molten flash cools, a homogenous weld joint is formed at or along the weld zone from the now intermixed materials of the welded components.
Spin-welding provides many advantages, such as relatively short cycle times, large batch sizes, and high overall process efficiency. Spin-welding also provides excellent repeatability when used in conjunction with precise process control methods, i.e., controlled material feed and/or spin rates, axial pressures, applied stroke, etc. However, the bonding strength and long term durability of a weld joint formed via a conventional spin-welding process may be less than optimal when used in conjunction with certain applications, and therefore the spin-welding process is generally restricted or limited to welding relatively small cylindrical parts of similar or like material in order to maximize the strength of the resultant weld joint.

Friction-welded assembly with interlocking feature and method for forming the assembly


The invention relates to a method for the maintenance of a plurality of processing stations (13) of a textile machine (10), whereby at least one first maintenance unit (14a-d) can travel alongside the processing stations (13) in order to service and/or control the processing stations (13). The maintenance unit (14a-d) may be moved into an optimized readiness position when none of the processing stations (13) needs to be serviced and/or controlled by the maintenance unit. Alternatively, the direction of travel in which the maintenance unit (14a-d) is to travel to the next processing station (13) in need of service is determined, whereby the direction of travel depends on the totality of processing stations in need of maintenance.

The present invention relates to a method for the maintenance of a plurality of processing stations of a textile machine, whereby at least one first maintenance unit can travel alongside the processing stations in order to service and/or control the processing stations.
In a known open-end spinning machine (DE 199 17 971 A1) a maintenance unit passes a plurality of spinning stations on one side of the open-end spinning machine and services the spinning station as needed. For this purpose, the maintenance unit patrols a row of spinning station along a guide rail. If the maintenance unit then encounters a spinning station that is signaling a defect, the maintenance unit attempts to put the spinning station back into operation. If this succeeds, the maintenance unit continues its travel in the original direction. If however the attempted maintenance fails several times, the affected spinning station is registered as not serviceable and the maintenance unit continues its travel and when it next passes the spinning station registered as non-serviceable that spinning station is no longer serviced by the maintenance unit. The spinning station's inability to be serviced is signaled to an operator so that he may carry out inspection tasks at the spinning station. In addition, a record is made if the maintenance unit is repeatedly unable to on several different spinning stations, it being then concluded that this is due to a malfunction of the maintenance unit. This malfunction is then also signaled and the inspection of the maintenance unit is carried out. By ignoring the spinning stations that cannot be serviced, the work efficiency of the maintenance unit is increased, since it no longer makes additional maintenance attempts at the non-serviceable spinning stations.

Method for the maintenance of a textile machine