Advance chromatin extraction enhances performance and productivity of cation exchange chromatography-based capture of Immunoglobulin G monoclonal antibodies

AbstractThe impact of host cell-derived chromatin was investigated on the performance and productivity of cation exchange chromatography as a method for capture-purification of an IgG monoclonal antibody. Cell culture supernatant was prepared for loading by titration to pH 6.0, dilution with water to a conductivity of 4mS/cm, then microfiltration to remove solids. DNA content was reduced 99% to 30ppm, histone host cell protein content by 76% to 6300ppm, non-histone host cell protein content by 15% to 321,000ppm, and aggregates from 33% to 15%. IgG recovery was 83%. An alternative preparation was performed, adding octanoic acid, allantoin, and electropositive particles to the harvest at pH 5.3, then removing solids. DNA content was reduced to<1 ppb, histones became undetectable, non-histones were reduced to 24,000ppm, and aggregates were reduced to 2.4%. IgG recovery was 95%. This treatment increased dynamic capacity (DBC) of cation exchange capture to 173g/L and enabled the column to reduce non-histone host proteins to 671ppm. Step recovery was 99%. A single multimodal polishing step further reduced them to 15ppm and aggregates to <0.1%. Overall process recovery was 89%. Productivity at feed stream IgG concentrations of 5–10g/L was roughly double the productivity of a same-size protein A column with a DBC of 55g/L

Chromatographic tools for optimization of IVT reaction and improving mRNA purification process

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown the promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. Typical mRNA production process involves three key steps: 1) plasmid DNA (pDNA) production in supercoiled (sc) isoform, linearization and purification, 2) in-vitro transcription (IVT) reaction and 3) mRNA purification. Here we present a chromatographic toolbox for integrated mRNA production from pDNA to mRNA purification, including in-process analytics. The pDNA purification approach presented here was designed to fit the specific requirements of mRNA vaccines. It integrates a linearisation step before polishing (removal of unwanted isoforms) of plasmid DNA. The polishing step after enzymatic linearisation, separates linear pDNA from enzyme and other unwanted products. Supporting in-process analytical tools are presented. IVT reaction monitoring with novel HPLC approaches includes CIMac PrimaS analysis of mRNA content as a function of time, with concomitant monitoring of NTP consumption. With information on NTPs, capping reagent and mRNA content, IVT reaction can be rapidly optimized for maximum productivity, in near real-time. Advantage of at-line monitoring is to prevent degradation of mRNA in IVT mixture which would occur after maximum productivity is reached. Purification of mRNA from IVT reaction mixture can be achieved using selective binding to polyA tail (using OligodT chromatography) or multimodal chromatography (PrimaS) which separates the ssRNA product from DNA template and IVT reaction mixture. Polishing approaches for final separation of ssRNA and dsRNA using hydrophobic interaction chromatography (HIC) and reverse-phase (RP) chromatography achieve high purity of final product. Supporting in-process analytical HPLC tools, including multimodal chromatography resin, facilitate a rapid read-out of mRNA concentration and purity profile

A tribute to Dr. Serge N. Timasheff, our mentor

63 p.-6 fig.Dr. Serge N. Timasheff, our mentor and friend, passed away in 2019. This article is a collection of tributes from his postdoctoral fellows, friends, and daughter, who all have been associated with or influenced by him or his research. Dr. Timasheff is a pioneer of research on thermodynamic linkage between ligand interaction and macromolecular reaction. We all learned a great deal from Dr. Timasheff, not only about science but also about life.Peer reviewe

Stability of IgMs at low pH used during purification and during storage

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Stability of IgMs at low pH used during purification and during storage

International audienc

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation

Increasing thermodynamic and kinetic stability during shear and thermal stress and long term storage of antibodies by excipients

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Immunoglobulins M Survive Low-pH Conditions Used for Virus Inactivation and for Elution from Bioaffinity Columns

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Structural Comparison of Cytotoxic versus Non-Cytotoxic Immunoglobulin M Antibodies Recognizing Undifferentiated Human Pluripotent Stem Cells

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