ABSTRACT
A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Pichia/metabolism , Recombinant Proteins/biosynthesis , Antibodies, Monoclonal/isolation & purification , Organisms, Genetically Modified , Pichia/genetics , Recombinant Proteins/isolation & purificationABSTRACT
In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.
Subject(s)
DNA Damage , Hepatocytes/drug effects , Mutagenicity Tests/methods , Mutagens/toxicity , Animals , Biological Assay , Cell Survival/drug effects , Cells, Cultured , Chlorophenols/toxicity , Chlorpheniramine/toxicity , DNA/analysis , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , In Vitro Techniques , Male , Nitrophenols/toxicity , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Toxicity Tests/methods , Toxicity Tests/standardsABSTRACT
The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.