Recovery and purification process development for monoclonal antibody production

Liu, Hui F.; Ma, Junfen; Winter, Charles M; Bayer, Robert

doi:10.4161/mabs.2.5.12645

Cited by 452 publications

(426 citation statements)

References 91 publications

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“…7 The impurity levels for host cell proteins by ELISA were < 50 ng/mg and residual DNA by qPCR was below the limit of quantitation of the assay. The AEX protein product was then diluted from 10 to 4.5 mg/mL with 1.4 M sodium sulfate, pH adjusted with 1 M Tris base (Tris (hydroxymethyl)aminomehtane), and 0.2 µm filtered to make the HIC load.…”

Section: Flowthrough Hic Purification With Real Time Mw By In-line Malsmentioning

confidence: 94%

“…6 Ion exchange chromatography (IEX), size-exclusion chromatography (SEC), and hydrophobic interaction chromatography (HIC) are all further polishing steps that can decrease aggregate levels. 7 These purification methods must be carefully designed because aggregate removal often comes at the expense of yield. Peak cutting or loading is often kept conservative to minimize the risk of aggregate breakthrough in the effluent.…”

mentioning

confidence: 99%

“…Peak cutting or loading is often kept conservative to minimize the risk of aggregate breakthrough in the effluent. 7,8 …”

mentioning

confidence: 99%

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Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Patel

Gospodarek

Larkin³

et al. 2018

mAbs

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For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

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Section: Flowthrough Hic Purification With Real Time Mw By In-line Malsmentioning

confidence: 94%

mentioning

confidence: 99%

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Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Patel

Gospodarek

Larkin³

et al. 2018

mAbs

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show abstract

“…Twofold serial dilutions (1:25 to 1:1,600) of sera were examined, and the inhibitory dose to achieve 50% neutralization (ID 50 ) was represented as the reciprocal value of the dilution (Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to GNA, recombinant protein A-or G-based chromatography media are readily available and widely used for commercial purification of monoclonal antibodies (49,50). Protein A medium exhibits high selectivity, a high flow rate, and a cost-effective binding capacity for antibodies and Fc-fusion proteins (dynamic capacity ranging from 15 to 50 mg of antibody/ml of resin) (51).…”

Section: Discussionmentioning

confidence: 99%

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

Logan

Law

Wong

et al. 2017

J Virol

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A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/ gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fctagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

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Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

Lü

et al. 2022

FEBS Open Bio

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Brazzein has excellent potential for use as a sweetener because of its high level of sweetening potency and stability against extreme temperature and pH. It is extracted from the tropical and difficult—to‐cultivate African plant Pentadiplandra brazzeana , which hampers its commercial viability. Here we report the mammary‐specific expression of wildtype or triple mutational (H31R/E36D/E41A) des ‐pGlu brazzeins in the milk of transgenic mice. Using enzyme‐linked immunoassay (ELISA), western blot, and sweetness intensity testing, we confirmed that the triple mutation made the des ‐pGlu brazzein molecule 10,000 times sweeter than sucrose in a weight base, even after 10 min of incubation at 100 °C; in addition, the triple mutant was also significantly sweeter than the wildtype des ‐pGlu brazzein. This study provides new insights for producing brazzein or brazzein‐sweetened milk from animals for use in food and healthcare applications.

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Recovery and purification process development for monoclonal antibody production

Cited by 452 publications

References 91 publications

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

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