Enhanced filtration performance using<scp>feed‐and‐bleed</scp>configuration for purification of antibody precipitates

Zhao, Li; Chen, Ting Hsi; Andini, Erha; Coffman, Jon; Przybycien, Todd M.; Zydney, Andrew L.

doi:10.1002/btpr.3082

Cited by 9 publications

(10 citation statements)

References 27 publications

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“…Precipitated and Protein A purified samples were further analyzed through LC‐MS to provide an estimated abundance level for the proteins of interest. The proteins listed in Table 4 are all serine hydrolases; a class of lipase enzymes previously reported to be related to PS‐80 degradation (Chiu et al, 2017; Dixit et al, 2016; Hall et al, 2016; Li et al, 2021). Additionally, Plbl2 has high immunogenicity risk for patients (Fischer et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

“…mAbs have been successfully precipitated using PEG and CaCl 2 , (Sommer et al, 2014) ZnCl 2 and PEG, (Li & Zydney, 2017) and elastin‐like polypeptides (ELP) among others (Mullerpatan et al, 2020; Swartz et al, 2018). However there have been significant challenges in recovering and washing the precipitated mAb through either centrifugation or tangential flow microfiltration (Li, Chen, et al, 2021). Alternatively, direct precipitation of impurities like HCP and DNA would be easier to implement into the existing purification process, without the need for washing and redissolution steps as required in target product precipitation (Li et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…mAbs have been successfully precipitated using PEG and CaCl 2 , (Sommer et al, 2014) ZnCl 2 and PEG, (Li & Zydney, 2017) and elastin-like polypeptides (ELP) among others (Mullerpatan et al, 2020;Swartz et al, 2018). However there have been significant challenges in recovering and washing the precipitated mAb through either centrifugation or tangential flow microfiltration (Li, Chen, et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

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Cationic polymer precipitation for enhanced impurity removal in downstream processing

Zhao

Martinez‐Fonts

Rauscher

et al. 2023

Biotech & Bioengineering

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Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cationic polymer precipitation for enhanced impurity removal in downstream processing

Zhao

Martinez‐Fonts

Rauscher

et al. 2023

Biotech & Bioengineering

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“…We developed a fully continuous capture process for monoclonal antibodies using continuous precipitation in tubular reactors coupled with continuous tangential flow filtration (TFF). [28][29][30][31][32] The antibody precipitated in a tubular reactor is continuously concentrated and washed by multiple stages of TFF, generating a continuous mass flow of product. A pool-less integrated perfusion-capture process over several days of process campaign could be operated.…”

Section: Introductionmentioning

confidence: 99%

Residence time distribution of continuous capture of recombinant antibody by precipitation and two stage tangential flow filtration

Recanati,

Lali,

De Mathia

et al. 2024

J of Chemical Tech & Biotech

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BACKGROUNDThe determination of the residence time distribution of an entire process or process step in integrated continuous biomanufacturing is required for process understanding, traceability and to efficiently handle process disturbances. When recombinant proteins such as monoclonal antibodies are captured by continuous precipitation and two stage filtration, the question arises whether simple salt is suitable as a tracer or whether a labeled protein is required.RESULTSWe have investigated the use of various inert tracers, NaNO3 due to its absorbance at UV 280 nm and fluorescently labeled or unlabeled antibodies to determine the RTD of the product at steady‐state and in the startup and shutdown phases. All tracers are suitable for measuring the RTD of the precipitated antibody for the individual unit operations. For the interconnected unit operations, if the product is concentrated, the RTD can be determined with labeled or unlabeled antibodies, because it is not possible to concentrate salts in a microfiltration unit. The duration of the start‐up and shut‐down phases depends on the concentration factor applied in two‐stage filtration, and the number of reactor volumes required to reach steady state corresponds to the concentration factor. Around 1.6 reactor volumes are required for the shutdown phase for 99% wash out.CONCLUSIONThe integrated process has a plug flow characteristic, which is advantageous as the process can react quickly when process deviations occur.This article is protected by copyright. All rights reserved.

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“…In this regard, more effort was carried on in recent years to develop entirely flow-through polishing of recombinant antibodies with the aim of generating a truly continuous mass flow of product and reducing process complexity [2][3][4][5][6]. The use of precipitation and filtration techniques for the capture and harvest of recombinant antibodies from clarified cell culture supernatant (CCCS) was also intensively explored over the last decade [7][8][9][10][11][12][13][14][15][16][17][18][19]. PEG precipitation principles are well understood [7,8,[20][21][22], however, integrated processes have not yet been established.…”

Section: Introductionmentioning

confidence: 99%

Integration of a perfusion reactor and continuous precipitation in an entirely membrane‐based process for antibody capture

Recanati,

Pappenreiter,

Gstoettner

et al. 2023

Engineering in Life Sciences

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Continuous precipitation coupled with continuous tangential flow filtration is a cost‐effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host‐cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab‐scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand‐alone capture and as an integrated perfusion‐capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow‐through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host‐cell proteins and host‐cell DNA by the combination of the precipitation‐based capture and the first polishing step.

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Enhanced filtration performance usingfeed‐and‐bleedconfiguration for purification of antibody precipitates

Cited by 9 publications

References 27 publications

Cationic polymer precipitation for enhanced impurity removal in downstream processing

Cationic polymer precipitation for enhanced impurity removal in downstream processing

Residence time distribution of continuous capture of recombinant antibody by precipitation and two stage tangential flow filtration

Integration of a perfusion reactor and continuous precipitation in an entirely membrane‐based process for antibody capture

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