Machine learning prediction of methionine and tryptophan photooxidation susceptibility.

Photooxidation of methionine (Met) and tryptophan (Trp) residues is common and includes major degradation pathways that often pose a serious threat to the success of therapeutic proteins. Oxidation impacts all steps of protein production, manufacturing, and shelf life. Prediction of oxidation liability as early as possible in development is important because many more candidate drugs are discovered than can be tested experimentally.

Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis.

Protein primary structure is a potential critical quality attribute for biotherapeutics. Identifying and characterizing any sequence variants present is essential for product development. A sequence variant ~11 kDa larger than the expected IgG mass was observed by size-exclusion chromatography and two-dimensional liquid chromatography coupled with online mass spectrometry.

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics.

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation.

High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system.

Biopharmaceutical protein manufacturing requires the highest producing cell lines to satisfy current multiple grams per liter requirements. Screening more clones increases the probability of identifying the high producers within the pool of available transfectant candidate cell lines. For the predominant industry mammalian host cell line, Chinese hamster ovary (CHO), traditional static-batch culture screening does not correlate with the suspension fed-batch culture used in manufacturing, and thus has little predictive utility.

Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing.

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines.

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality.

The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low-density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells.

Effectiveness of mouse minute virus inactivation by high temperature short time treatment technology: a statistical assessment.

Viral contamination of mammalian cell cultures in GMP manufacturing facility represents a serious safety threat to biopharmaceutical industry. Such adverse events usually require facility shutdown for cleaning/decontamination, and thus result in significant loss of production and/or delay of product development. High temperature short time (HTST) treatment of culture media has been considered as an effective method to protect GMP facilities from viral contaminations.

A Bayesian Approach for Multiple Response Surface Optimization in the Presence of Noise Variables.

An approach for the multiple response robust parameter design problem based on a methodology by Peterson (2000) is presented. The approach is Bayesian, and consists of maximizing the posterior predictive probability that the process satisfies a set of constraints on the responses. In order to find a solution robust to variation in the noise variables, the predictive density is integrated not only with respect to the response variables but also with respect to the assumed distribution of the noise variables.