Monoclonal antibody production with on-line harvesting and process monitoring

Zhang, Jifeng; Zhou, Hong; Ji, Zhisong; Regnier, Fred E.

doi:10.1016/s0378-4347(97)00472-6

Cited by 14 publications

(4 citation statements)

References 9 publications

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“…MAb 2C3 is a murine IgG1 antibody that binds to the 14.6-kDa H.8 protein of pathogenic Neisseria ( 33 , 34 ). For bactericidal studies, MAb 6E4 was purified over a protein G column using standard elution techniques ( 35 ).…”

Section: Methodsmentioning

confidence: 99%

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Apicella

Coffin

Ketterer

et al. 2018

mBio

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Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.

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Section: Methodsmentioning

confidence: 99%

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Apicella

Coffin

Ketterer

et al. 2018

mBio

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“…More specifically, quality attributes such as the glycosylation profile, truncation and incorrect assembly of mAb subunits, oxidation, deamidation and glycation are profoundly affected by the cell culture conditions, and therefore need proper control. [3][4][5][6][7][8][9][10][11][12] Simultaneous monitoring of these attributes may thus enable instant tuning of process parameters, e.g., temperature, pH value, and cultivation time, to achieve the desired product profile. 1,13 In IgG1-type mAbs, N-glycosylation represents a critical quality attribute (CQA) because it affects the safety and efficacy of the therapeutic protein.…”

Section: Introductionmentioning

confidence: 99%

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study

Regl

Wohlschlager

Esser‐Skala

et al. 2019

mAbs

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Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N -glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N -glycans enables determination of mAb glycation levels, which are subsequently considered in relative N -glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.

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“…Moreover, relatively large amounts of buffer are required, which adds further to the waste load. Furthermore, results may be subject to interpretation and affected by interactions between the mAb and the matrix that lead to peak broadening 5, 7–9. Capillary electrophoresis systems may also be used for automated mAb analysis.…”

Section: Introductionmentioning

confidence: 99%

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy

Capito

Skudas

Stanislawski

et al. 2012

Biotechnology Progress

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Production of recombinant proteins, e.g. antibodies, requires constant real-time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers. To date, this monitoring of mammalian cell culture-based processes is done using laborious and time consuming enzyme-linked immunosorbent assays (ELISA), two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis, and chromatography-based systems. Measurements are usually performed off-line, requiring regular sample withdrawal associated with increased contamination risk. As information is obtained retrospectively, the reaction time to adapt to process changes is too long, leading to lower yield and higher costs. To address the resulting demand for continuous online-monitoring systems, we present a feasibility study using attenuated total reflection spectroscopy (ATR) to monitor mAb and HCP levels of NS0 cell culture in situ, taking matrix effects into account. Fifty-six NS0 cell culture samples were treated with polyelectrolytes for semi-selective protein precipitation. Additionally, part of the samples was subjected to filtration prior to analysis, to change the background matrix and evaluate effects on chemometric quantification models. General models to quantify HCP and mAb in both filtered and unfiltered matrix showed lower prediction accuracy compared to models designed for a specific matrix. HCP quantification in the range of 2,000-55,000 ng mL(-1) using specific models was accurate for most samples, with results within the accepted limit of an ELISA assay. In contrast, mAb prediction was less accurate, predicting mAb in the range of 0.2-1.7 g L(-1) . As some samples deviated substantially from reference values, further investigations elucidating the suitability of ATR for monitoring are required.

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Monoclonal antibody production with on-line harvesting and process monitoring

Cited by 14 publications

References 9 publications

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy

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