Characterization of a cathepsin D protease from CHO cell-free medium and mitigation of its impact on the stability of a recombinant therapeutic protein.

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH.

Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer.

Monoclonal antibodies have been used increasingly as therapeutic agents to target various diseases. Although most monoclonal antibodies have only one N-linked glycosylation site in the Fc region, N-linked glycosylation sites in the Fab region have also been observed. Because glycosylation of a monoclonal antibody can have a significant impact on its effector function, efficacy, clearance, and immunogenicity, it is essential to assess the glycosylation profile during cell line and clone selection studies and to assess the impact of cell culture conditions on the glycoform distribution during process optimization studies to ensure that the antibody is being produced with appropriate and consistent glycosylation.

Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals.

Therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. The use of mass spectrometry (MS) for the evaluation of recombinant protein sequence and structure provides detailed information regarding amino acid modifications and sequence alterations that have the potential to affect the safety and activity of therapeutic protein products. General MS approaches for the characterization of recombinant therapeutic protein products will be reviewed with particular attention given to the standard MS tools available in most biotechnology laboratories.

Detailed structural analysis of amyloidogenic wild-type transthyretin using a novel purification strategy and mass spectrometry.

Wild-type transthyretin (TTR), normally a soluble plasma-circulating protein, can be amyloidogenic, i.e., form tissue-deposited fibrillar material in the extracellular matrix of various organs throughout the body.

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma.

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry.

In vitro and in vivo interactions of homocysteine with human plasma transthyretin.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer's disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease.

D18G transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?

Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date.

Identification of a novel transthyretin Thr59Lys/Arg104His. A case of compound heterozygosity in a Chinese patient diagnosed with familial transthyretin amyloidosis.

Transthyretin (TTR) is a 127-amino acid residue protein synthesized mainly in the liver and in several minor sites, including the choroid plexus and the eye. In plasma, TTR circulates as a homotetramer and transports the hormone thyroxine and the retinol-binding protein-vitamin A complex. It is hypothesized that amino acid substitutions in TTR destabilize the tetramer by causing each subunit toform intermediates that may self-associate into amyloid fibrils.

Molecular organization of amyloid protofilament-like assembly of betabellin 15D: helical array of beta-sandwiches.

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers.

Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis.

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils.