Bayer Corporation

Joseph A Buettner

George A Baumbach

Christopher A Dadd

David J Hammond

June P Davis

Ruben Guillermo Carbonell

Li Ang Chen

Ruben C Carbonell

Kristine Mondorf

Peptides that have domains that bind to recombinant factor VIII (rFVIII) are disclosed. A method of rFVIII binding assay using the peptides deduced from a combinatorial library in a filtration plate process is described. A method of using peptides having these available binding domains in an affinity chromatography process to purify factor VIII is also taught.

Recombinant factor VIII binding peptides

The invention is a method of purifying Factor VIII comprising the steps of contacting a solution containing Factor VIII with a Factor VIII-binding substrate under conditions sufficient to bind Factor VIII to the substrate, wherein the Factor VIII-binding substrate comprises one or more peptides bound to the substrate, wherein the peptides are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and then eluting the bound Factor VIII.

Method of purifying human factor VIII

Peptides which bind Factor VIII are disclosed. The peptides have available Factor VIII binding domains having a Trp-His-Tyr-Tyr-His-Gly, His-Ile-Gln-His-Tyr-His, or His-Gln-Tyr-Gly-Tyr-His sequence. Peptides having at least one of these Factor VIII binding domains are immobilized upon a chromatographic substrate in a preferred embodiment of the invention. This preferred embodiment is useful in a chromatography process to purify human Factor VIII.

Peptide ligands for affinity purification of human Factor VIII

Ligands that interact with a target can be more easily identified if false positive interactions (either specific or non-specific) from the detecting system are differentiated from the target-specific interaction. An improved method of identifying peptides which bind with a target protein is presented. The steps are: binding a random library of peptides to a support material, allowing detection reagents to contact the peptides and the support material then identifying these interactions, then allowing the target protein to selectively bind to the peptides, allowing detection reagents to contact the bound target protein, and characterizing the peptide bound to the identified support material. Interaction of a ligand or the support material with the detection reagents will cause a distinct color change which distinguishes those ligands which selectively bind to target protein. The characterized peptide can then be used in affinity purification of the target protein. In one embodiment, automation of the assay is demonstrated by flowing all immunoreagents through the beads in a column format ensuring highly efficient washing. In the preferred embodiment, a resin for peptide synthesis which is hydrophilic, contains spacers and may exhibit less nonspecific background than other resins permits synthesis and direct evaluation of combinatorial peptide libraries for binding to target proteins is utilized. Examples for the use of this new resin and methodology for identifying peptide-ligands for purification of proteins are presented.

Screening of combinatorial peptide libraries for selection of peptide

Peptide ligands which bind to thrombin and prothrombin are disclosed. The sequences of these peptides are Gln-Leu-Trp-Gly-Ser-His, Arg-Gln-Leu-Trp-Gly-Ser-His, His-Gln-Leu-Trp-Gly-Ser-His, and Tyr-Phe-Pro-Gly-Pro-Tyr-Leu. The preferred peptides have the sequence Gln-Leu-Trp-Gly-Ser-His or Tyr-Phe-Pro-Gly-Pro-Tyr-Leu. A method of using these peptides in an affinity chromatography process to purify thrombin is described.

Peptides which bind to prothrombin and thrombin

Peptides which bind to fibrinogen are disclosed. These peptides have available fibrinogen binding domains having a Trp-Gln-Glu-His-Tyr-Asn, Trp-Gln-Glu-Thr-TyrGln,or Tyr-Glu-Asn-Tyr-Gly-Tyr sequence. Peptides containing at least one of these fibrinogen binding domains are immobilized upon a chromatographic substrate in a preferred embodiment of the invention. This preferred embodiment is useful in an affinity chromatography process to purify fibrinogen.

Peptide ligands for affinity purification of fibrinogen

Peptides which bind to fibrinogen are disclosed. These peptides have available fibrinogen binding domains having a Phe-Leu-Leu-Val or Leu-Leu-Val-Pro sequence. The preferred peptide is a 6-mer having the sequence Phe-Leu-Leu-Val-Pro-Leu (SEQ ID NO:1). A method of using these peptides in an affinity chromatography process to purify fibrinogen is described.

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Raleigh, NC, United States of America

Joseph A Buettner