Creation and filtering of a recurrent spectral library of CHO cell metabolites and media components.

This paper reports the first implementation of a new type of mass spectral library for the analysis of Chinese hamster ovary (CHO) cell metabolites that allows users to quickly identify most compounds in any complex metabolite sample. We also describe an annotation methodology developed to filter out artifacts and low-quality spectra from recurrent unidentified spectra of metabolites. CHO cells are commonly used to produce biological therapeutics. Metabolic profiles of CHO cells and media can be used to monitor process variability and look for markers that discriminate between batches of product. We have created a comprehensive library of both identified and unidentified metabolites derived from CHO cells that can be used in conjunction with tandem mass spectrometry to identify metabolites. In addition, we present a workflow that can be used for assigning confidence to a NIST MS/MS Library search match based on prior probability of general utility. The goal of our work is to annotate and identify (when possible), all liquid chromatography-mass spectrometry generated metabolite ions as well as create automatable library building and identification pipelines for use by others in the field.

Heterologous recombinant expression of non-originator NISTmAb.

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.

Protein labeling in Escherichia coli with (2)H, (13)C, and (15)N.

A number of structural biology techniques such as nuclear magnetic resonance spectroscopy and small-angle neutron scattering can be performed with proteins with nuclei at natural isotope abundance. However, the use of proteins labeled with stable isotopes ((2)H, (13)C, and (15)N) enables greater experimental flexibility. In this chapter, several methods for uniform and fractional protein labeling with stable isotopes using Escherichia coli in a defined media are described. The methods described can be used for labeling with single or multiple isotopes.