Improved processes for the preparation of tubulysin compounds, tubulysin drug linker compounds, and their intermediates are disclosed.

The present invention provides a novel process of PEGylation and purification of the PEGylated therapeutic proteins. The developed PEGylation protocol provides high yield of PEGylated recombinant proteins (with % mono-PEG conversion of ≥70%) with a much faster reaction completion time indicating high productivity and fast kinetics. Thereafter a displacement mode cation exchange chromatography (CEX) is utilized which is able to remove all the multi-PEGylated impurities in the loading flow-through, simplifying the overall purification process. The novel PEGylation and purification process can be integrated to any existing manufacturing process to offer an end-to-end integrated assembly from inclusion bodies to purified PEGylated product with higher productivity and improved resin utilization.

Embodiments of the present disclosure pertain to methods of conjugating a molecule to a polypeptide by (1) modifying one or more thiol residues on the polypeptide, where the modifying includes cyanylation of the one or more thiol residues; and (2) associating the polypeptide with the molecule, where the associating results in the conjugation of the molecule to the polypeptide through a reaction between a nucleophilic moiety on the molecule and the one or more modified thiol residues. The cyanylation may include attachment of cyano groups to sulfur atoms of the one or more thiol residues to form thiocyanato groups that undergo reversible intramolecular addition with a nearby N-amide group to generate a 1-acyl-2-iminothiazolidine intermediate. Thereafter, the nucleophilic moiety on the molecule reacts with the 1-acyl-2-iminothiazolidine intermediate to replace 2-iminothiazolidine in a nucleophilic acyl substitution reaction and result in the conjugation of the molecule to the polypeptide.

The invention relates to materials and methods of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a therapeutic protein comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation. More specifically, the present invention relates to the aforementioned materials and methods wherein the water soluble polymer contains an active aminooxy group and wherein an oxime or hydrazone linkage is formed between the oxidized carbohydrate moiety and the active aminooxy group on the water soluble polymer, and wherein the conjugation is carried out in the presence of a nucleophilic catalyst.

The present invention relates to cell culture media comprising 5-Thio-L-fucose and the use of 5-Thio-L-fucose for modulating the glycosylation profile of antibodies or other Fc containing proteins and thereby modulating the binding affinity of said proteins.

It was found that a peptide compound that has an N-substituted-α,α-disubstituted amino acid residue at the N-terminus and containing a dipeptide residue in which the N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked together, can be efficiently produced by linking an N-unsubstituted-α,α-disubstituted amino acid whose amino group is protected with an electron-withdrawing protecting group to an N-substituted amino acid or a peptide compound having an N-substituted amino acid residue at the N-terminus, and then allowing a substituent-introducing agent to act in the presence of a specific base to selectively introduce a substituent to the amino group at the N-terminus.

The present invention relates generally to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a spacer containing ((2-oxoethyl)thio)). In an aspect the invention provides oxo-eT linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a ((2-oxoethyl)thio) spacer having the formula (I): ##STR00001## wherein: A is a group (C═X).sub.m wherein X is S or O and m is 0 or 1; B is a bond, O, or CH.sub.2; and when m is 0, B can also be (C═O); R is a C.sub.2-C.sub.16 alkylene, C.sub.2-C.sub.16 heteroakylene, NH—C(═O)—C.sub.2-C.sub.16 alkylene, or NH—C(═O)—C.sub.2-C.sub.16 heteroakylene, wherein said alkylene and heteroalkylene are optionally substituted by 1, 2 or 3 groups independently selected from COOR′ where R′ is selected from H, methyl, ethyl or propyl. The invention further relates to immunogenic compositions comprising such glycoconjugates, and to methods for the preparation and use of such glycoconjugates and immunogenic compositions.

The present invention relates to a facile acylation process for preparation of N-Substituted peptide and proteins. More specifically, the invention relates to acylating a peptide or a protein with deprotected acylating agent.

Disclosed herein, in some aspects, are synthetic secretion signal peptides. Also disclosed are nucleic acid molecules encoding such signal peptides, in some cases operably linked to a protein coding sequence, as well as cells comprising such nucleic acid molecules. Further disclosed are methods for secreting a polypeptide comprising expressing in a cell a signal peptide of the disclosure linked to the polypeptide. Certain aspects include proteins (e.g., human milk proteins) produced by such methods, as well as compositions comprising such proteins.

Site-specific modifications of proteins are desirable in biotechnological applications such as biopharmaceuticals, immunotherapy, vaccines, and are useful in chemical biology. Gluconoylation is a non-enzymatic, covalent, post-translational modification commonly observed on N-terminal His-Tags bearing proteins. We synthesized glucono-1,5-lactone derivatives, including azido variants for selective acylation. High yield acylation is achieved by simply mixing derivatives with target protein amidst diverse conditions of temperatures, aqueous buffers, excipients, or complex cell lysate.