2-D DIGE to expedite downstream process development for human monoclonal antibody purification

Grzeskowiak, Julita K.; Tscheliessnig, Anne; Toh, Poh Choo; Chusainow, Janet; Lee, Yih Yean; Wong, Niki S.C.; Jungbauer, Alois

doi:10.1016/j.pep.2009.01.007

Cited by 46 publications

(47 citation statements)

References 33 publications

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“…This larger difference can also be visually seen by examining the gel image shown in Figure 5. A similar effect of cell viability on HCP population has also been noted by Grzeskowiak et al (2009).…”

Section: Effect Of Cell Viability At Harvestsupporting

confidence: 76%

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Jin

Szapiel

Zhang

et al. 2009

Biotech & Bioengineering

151

165

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Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.

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Section: Effect Of Cell Viability At Harvestsupporting

confidence: 76%

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Jin

Szapiel

Zhang

et al. 2009

Biotech & Bioengineering

151

165

View full text Add to dashboard Cite

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“…Precipitation by the commercially available 2D clean-up kit was performed according to manufacturer protocol as described previously [6]. …”

Section: Methodsmentioning

confidence: 99%

“…For example, the extracellular CHO HCP composition has been shown to vary minimally with changes in a variety of cell culture conditions, such as composition of media and temperature shift [4], and extracellular CHO autocrine growth factors have been identified to optimize the composition of serum-free media [5]. Cell culture viability has been demonstrated to impact significantly the extracellular proteome by introduction of intracellular CHO HCPs at decreased viability [4, 6, 7]; however, even with viability greater than 90%, extracellular proteome changes were observed over the initial six days of cell culture [8]. In downstream purification, proteomic techniques have been used to demonstrate the impact of primary recovery operations on the extracellular CHO HCP profile [9], show impurity clearance across protein A and ion-exchange chromatography operations [6], and define operating conditions for a variety of mixed-mode chromatography resins [10].…”

Section: Introductionmentioning

confidence: 99%

“…Cell culture viability has been demonstrated to impact significantly the extracellular proteome by introduction of intracellular CHO HCPs at decreased viability [4, 6, 7]; however, even with viability greater than 90%, extracellular proteome changes were observed over the initial six days of cell culture [8]. In downstream purification, proteomic techniques have been used to demonstrate the impact of primary recovery operations on the extracellular CHO HCP profile [9], show impurity clearance across protein A and ion-exchange chromatography operations [6], and define operating conditions for a variety of mixed-mode chromatography resins [10]. Additionally, CHO HCPs are highly conserved across different cell lines [4, 6, 11], and non-recombinant protein producing (null) CHO cells have demonstrated CHO HCP compositions equivalent to those of cell lines producing therapeutic proteins [4, 6, 7].…”

Section: Introductionmentioning

confidence: 99%

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Recovery of Chinese hamster ovary host cell proteins for proteomic analysis

Valente

Schaefer

Kempton

et al. 2013

Biotechnology Journal

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Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. We developed optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of the precipitants by a design of experiments approach. Because total protein recovery cannot describe physicochemical characteristics of proteins or detect non-protein agents, which may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.

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“…Therefore, 2-D DIGE is a more accurate quantitative and qualitative method than 2-DE (37) and thus has been applied in proteomic studies of human diseases including cancer (38)(39)(40), autoimmune uveitis (41), chronic inflammatory demyelinating polyneuropathy (42) and psychiatric disease (43). In addition, 2-D DIGE has also been applied for purification of human monoclonal antibodies (44) and determination of drug resistance (45).…”

Section: Introductionmentioning

confidence: 99%

Histidine Decarboxylase Is Identified as a Potential Biomarker of Intestinal Mucosal Injury in Patients with Acute Intestinal Obstruction

Yang

Zhāng

et al. 2011

Mol Med

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Various biomarkers currently used for the diagnosis of intestinal mucosal injury (IMI) in patients with acute intestinal obstruction have low sensitivity and specificity. In the present study, IMI, as indicated by the impaired expression of tight junction proteins, including zonula occludens-1, occludin and claudin-1, and inflammation were determined in colonic tissues of patients with 45 strangulated intestinal obstruction (STR-IO) and the adjacent "normal" colonic tissues of 35 patients with colon cancers by quantitative real-time polymerase chain reaction (QRT-PCR), Western blotting, immunohistochemistry and histological examination, respectively. Then, two-dimensional fluorescent difference gel electrophoresis coupled with linear trap quadrupole mass spectrometry was used to screen for potential biomarkers of IMI in the serum samples of 10 STR-IO, 10 simple intestinal obstruction (SIM-IO) and 10 normal healthy controls. A total of 35 protein spots were differentially expressed among the serum samples, and six of the proteins were identified as potential biomarkers. Among the six proteins, histidine decarboxylase (HDC) and ceruloplasmin (CP) were elevated significantly in patients with STR-IO, compared with patients with SIM-IO and healthy controls. Thus, HDC and CP were further validated by QRT-PCR, Western blotting, immunohistochemistry and enzyme-linked immunosorbent assay, respectively, in colonic tissues, serum and urine samples. Finally, the receiver operating characteristic curves were used to show the area under the curves of HDC, CP and several established biomarkers, followed by the determination of the appropriate cutoff values and their sensitivities and specificities. It was shown that for serum and urine, HDC levels achieved sensitivities and specificities compatible to or even greater than those of established biomarkers for the diagnosis of IMI in patients with acute intestinal obstruction, although further validation in a larger cohort is required.

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2-D DIGE to expedite downstream process development for human monoclonal antibody purification

Cited by 46 publications

References 33 publications

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Recovery of Chinese hamster ovary host cell proteins for proteomic analysis

Histidine Decarboxylase Is Identified as a Potential Biomarker of Intestinal Mucosal Injury in Patients with Acute Intestinal Obstruction

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