https://www.cell.com/trends/biotechnology/pdf/S0167-7799(04)00234-3.pdf

High-throughput cell-free systems for synthesis of functionally active proteins

Oxidation is a common degradation pathway that affects therapeutic proteins and peptides during production, purification, formulation, transportation, storage and handling of solid and liquid preparations. In the present work we review the scientific literature about structural and biological consequences of protein/peptide oxidation. Representative examples are discussed of specific products whose oxidation has been recently studied, including monoclonal antibodies, calcitonin, granulocyte colony-stimulating factor, growth hormone, insulin, interferon alpha and beta, oxytocin and parathyroid hormone. These examples illustrate that oxidation often leads to modifications of higher-order structures, including aggregate induction, and can generate products that are pharmacokinetically different, biologically less active and/or potentially more immunogenic than their native counterpart. It is therefore crucially important during the pharmaceutical development of therapeutic proteins and peptides to comprehensively characterize oxidation products and evaluate the impact of oxidation-induced structural modifications on the biological properties of the drug.

Oxidation of Therapeutic Proteins and Peptides: Structural and Biological Consequences

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

https://www.tandfonline.com/doi/pdf/10.4161/mabs.27876

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes

The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences.

Rationally designed, synthetic antibody libraries and uses therefor

In one aspect, biocompatible matrices such as sol-gels encapsulating a reaction center may be administered to a subject for conversion of prodrugs into biologically active agents. In certain embodiments, the biocompatible matrices of the present invention are sol-gels. In one embodiment, the enzyme L-amino acid decarboxylase is encapsulated and implanted in the brain to convert L-dopa to dopamine for treatment of Parkinson's disease.

Matrices for drug delivery and methods for making and using the same

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.

/pdf/identification-of-potential-sites-for-tryptophan-oxidation-3zj6f6hoo7.pdf

Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS.

The invention provides the means to enhance in E.coli-based expression systems the formation of fusion polypeptides containing as an N-terminal tag a biotinylation polypeptide. By way of specifically exchanging in the nucleic acid sequence encoding the biotinylation polypeptide nucleotides at 11 discrete positions enhances the formation of the total fusion polypeptide by at least 40%.

Enhanced expression of fusion polypeptides with a biotinylation tag

Controlled polysorbate 20 hydrolysis - A new approach to assess the impact of polysorbate 20 degradation on biopharmaceutical product quality in shortened time.

An enzyme selected from penicillin-G amidase, glutaryl-7-ACA acylase and D-amino acid oxidase is immobilized by covalent bonding on an aminofunctional organosiloxane polymer carrier to provide an immobilized enzyme having a specific volume activity of at least 100 U/g wet carrier. Preferably, the carrier has an average diameter or 0.01 to 3 mm and is essentially spherical. Covalent bonding is accomplished by activating amino groups on the carrier with a dialdehyde and reacting the activated groups with reactive groups on the enzyme. An amount of enzyme is immobilized to provide a weight ratio of enzyme to carrier of 1 to 300 mg protein per g wet carrier.

Immobilization of penicillin G amidase, glutaryl-7-ACA acylase or D-aminoacid oxidase on an aminofunctional organosiloxane polymer carrier

Aim: Host cell proteins represent a major process-related impurity in recombinant biotherapeutics. Present work reports about a fully automated platform based on a cobas instrument (Roche Diagnostic GmbH, Mannheim, Germany) allowing high-throughput determination of host cell proteins. Materials & methods: The instrument combines automated sample preparation with electrochemiluminescence-based detection technology, facilitating highly sensitive and accurate detection. Results: Our data shows that the assay performs readily comparable to conventional ELISAs, but outperforms in speed, sample throughput and by its superior linear range. Comparison of ELISA and electrochemiluminescence immunoassay validation results from more than ten independent validations of clinical product testing methods supports our comparability claim. Conclusion: The electrochemiluminescence immunoassay represents a milestone for bioprocess impurity testing and abolishes the sample throughput limitations posed by conventional ELISA.

/pdf/getting-cho-host-cell-protein-analysis-up-to-speed-1hp4shaqpj.pdf

Frank Wedekind

Papers

Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS.

Enhanced expression of fusion polypeptides with a biotinylation tag

Controlled polysorbate 20 hydrolysis - A new approach to assess the impact of polysorbate 20 degradation on biopharmaceutical product quality in shortened time.

Immobilization of penicillin G amidase, glutaryl-7-ACA acylase or D-aminoacid oxidase on an aminofunctional organosiloxane polymer carrier

Getting CHO host cell protein analysis up to speed