Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants

Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

doi:10.1007/s00216-015-8672-8

Cited by 28 publications

(28 citation statements)

References 17 publications

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“…The selected PLSR models were able to predict the aggregate concentrations based on the recorded EEMs. Furthermore, predicted and measured concentrations were closely aligned in this work ( Figure 3) and in the previously published work of Paul et al (2015) [16]. Therefore, it can be concluded that an interaction of dye and media proteins was either compensated in the PLSR model or it was rather negligible.…”

Section: Extrinsic Dye-related Emission Signalssupporting

confidence: 88%

“…In contrast, ThT was identified to be more toxic than ANS or Bis-ANS. Therefore ThT was only used in a rather low concentration compared to Paul et al (2015) [16], which resulted in low fluorescence signal intensities in the area of λ ex 370-450 nm and λ em 410-590 nm. In contrast, the same Bis-ANS concentration generated higher fluorescence intensities in the same EEM area.…”

Section: Chemometric Modelingmentioning

confidence: 99%

“…It was shown by various groups that mAb aggregation can already occur during USP, since aggregation can be influenced by medium components, ionic strength, pH control, and temperature shift [13][14][15]. Paul et al (2015) analyzed mAb aggregates directly in CHO cell culture supernatant via size exclusion chromatography, and reported roughly 77% aggregated mAb [16]. A high aggregate content during USP might eventually reduce the overall yield and increase the burden on downstream processing.…”

Section: Introductionmentioning

confidence: 99%

“…Extrinsic dyes are usually applied as fluorescent markers in tissue sections, protein purification, and formulation development [20][21][22][23]. However, Paul et al (2015) have already applied ANS and Bis-ANS for the detection of aggregated mAb in cell-free CHO culture supernatant. They reported that specific regions of the recorded excitation emission matrices (EEM) showed increased fluorescence signals which were directly related to increased aggregate concentrations [16].…”

Section: Introductionmentioning

confidence: 99%

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Hybrid Modelling and Multi- Parametric Control of Bioprocesses

2018

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Section: Extrinsic Dye-related Emission Signalssupporting

confidence: 88%

Section: Chemometric Modelingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

2018

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“…Using our recently developed method for high-throughput analysis of soluble aggregates (Paul et al, 2015) and a method to quantify mAb aggregate particles (Paul et al, 2017), we investigated the impact of the process relevant factors temperature, osmolality, agitation, antifoam, and VPA on the cell concentration and formation of mAb aggregates in mAb-producing CHO cell cultures. Using our recently developed method for high-throughput analysis of soluble aggregates (Paul et al, 2015) and a method to quantify mAb aggregate particles (Paul et al, 2017), we investigated the impact of the process relevant factors temperature, osmolality, agitation, antifoam, and VPA on the cell concentration and formation of mAb aggregates in mAb-producing CHO cell cultures.…”

mentioning

confidence: 99%

Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture

Paul

Handrick

Ebert

et al. 2018

Biotech & Bioengineering

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Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture.

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Inhibition of VEGF‐dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a

et al. 2018

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The C‐type lectin‐like domain of CLEC14a (CLEC14a‐C‐type lectin‐like domain [CTLD]) is a key domain that mediates endothelial cell–cell contacts in angiogenesis. However, the role of CLEC14a‐CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity‐determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti‐angiogenic human monoclonal antibody that specifically targets CLEC14a‐CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a‐CTLD‐mediated endothelial cell–cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)‐dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC‐1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab‐adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a‐CTLD has the potential to suppress VEGF‐dependent angiogenesis and tumor angiogenesis and that CLEC14a‐CTLD may be a novel anti‐angiogenic target for VEGF‐dependent angiogenesis and tumor angiogenesis.

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Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants

Cited by 28 publications

References 17 publications

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture

Inhibition of VEGF‐dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a

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