Characterization of Sample Loss Caused by Competitive Adsorption of Proteins in Vials Using Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Ranade, Ameya V; Mukhtarov, Rustam; Liu, Ken Jia An; Behrner, Melissa A.; Sun, Bingyun

doi:10.1021/acs.langmuir.8b04281

Cited by 9 publications

(12 citation statements)

References 66 publications

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“…We combined the lab‐scale recirculation experiment with a rinse‐stripping protocol adapted from Lee et al (2011) and Ranade et al (2019). Before each experiment (10 total), the cRPP was cleaned in an ultrasonic bath and then rinsed extensively with Milli‐Q water.…”

Section: Methodsmentioning

confidence: 99%

Adsorbed protein film on pump surfaces leads to particle formation during fill‐finish manufacturing

Roffi

Pantazis

2021

Biotech & Bioengineering

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During fill-finish manufacturing, therapeutic proteins may aggregate or form subvisible particles in response to the physical stresses encountered within filling pumps. Understanding and quantitating this risk is important since filling may be the last unit operation before the patient receives their dose. We studied particle formation from lab-scale to manufacturing-scale using sensitive and robust protein formulations. Filling experiments with a ceramic rotary piston pump were integrated with a rinse-stripping method to investigate the relationship between protein adsorption and particle formation. For a sensitive protein, multilayer film formation on the piston surface correlated with high levels of subvisible particles in solution. For a robust protein formulation, adsorption and subvisible particle formation were minimal. These results support an aggregation mechanism that is initiated by adsorption to pump surfaces and propagated by mechanical and/or hydrodynamic disruption of the film. The elemental analysis confirmed that ceramic wear debris remained at trace levels and did not contribute appreciably to protein aggregation.

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Section: Methodsmentioning

confidence: 99%

Adsorbed protein film on pump surfaces leads to particle formation during fill‐finish manufacturing

Roffi

Pantazis

2021

Biotech & Bioengineering

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“…We used a published trypsin digestion protocol [33,34] and monitored digestion efficiency overnight and at 24 and 48 h. The SDS-PAGE gel results of CHO cell lysate digestion at 24 and 48 h are shown in Figure 2. No obvious difference was observed among all the time points chosen.…”

Section: Trypsin Digestionmentioning

confidence: 99%

“…Control Chinese hamster ovary (CHO) cells were maintained in sterile F12 (Ham's) nutrient medium, pH 7.4 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS (Life Technologies) and maintained in a humidified environment at 37 • C with 5% CO 2 (Symphony 5.3A, VWR, Radnor, PA, USA) until 90% confluency. The CHO cells were transiently cotransfected with human cDNA encoding the Nav1.5 α-subunit, the β1-subunit, and eGFP following the PolyFect (Qiagen, Germantown, MD, USA) transfection protocol [33]. Transfected cells were grown in filtered sterile F12 (Ham's) nutrient medium, pH 7.4 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% FBS and maintained in a humidified environment at 37 • C with 5% CO 2 [46].…”

Section: Cell Culture and Harvestingmentioning

confidence: 99%

Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry

Adams

Fouda

et al. 2022

IJMS

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Nav1.5 is the pore forming α-subunit of the cardiac voltage-gated sodium channel that initiates cardiac action potential and regulates the human heartbeat. A normal level of Nav1.5 is crucial to cardiac function and health. Over- or under-expression of Nav1.5 can cause various cardiac diseases ranging from short PR intervals to Brugada syndromes. An assay that can directly quantify the protein amount in biological samples would be a priori to accurately diagnose and treat Nav1.5-associated cardiac diseases. Due to its large size (>200 KD), multipass transmembrane domains (24 transmembrane passes), and heavy modifications, Nav1.5 poses special quantitation challenges. To date, only the relative quantities of this protein have been measured in biological samples. Here, we describe the first targeted and mass spectrometry (MS)-based quantitative assay that can provide the copy numbers of Nav1.5 in cells with a well-defined lower limit of quantification (LLOQ) and precision. Applying the developed assay, we successfully quantified transiently expressed Nav1.5 in as few as 1.5 million Chinese hamster ovary (CHO) cells. The obtained quantity was 3 ± 2 fmol on the column and 3 ± 2 × 104 copies/cell. To our knowledge, this is the first absolute quantity of Nav1.5 measured in a biological sample.

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“…A recent 2019 publication by Ranade et al . commented on the use of stripping agents to remove tightly adhered milk proteins from sample vials, particularly polypropylene vials 6 . Interestingly, they found that the metal chelator EDTA enhanced the surface adsorption of milk proteins.…”

Section: Introductionmentioning

confidence: 99%

“…This strategy proved very effective for a 27‐residue commercial peptide called Vn96 but was ineffective for the quantitative analysis of the intact proteins apomyoglobin and carbonic anhydrase. Our final strategy combined the use of low‐bind vials with the addition of an active‐site blocking agent or adsorption competitor 6 . In this case, we used the intact protein carbonic anhydrase at a concentration of 10 µg/mL as a blocker for apomyoglobin and the polypeptide ubiquitin at a concentration of 10 µg/mL as a blocker for carbonic anhydrase.…”

Section: Introductionmentioning

confidence: 99%

Improved intact peptide and protein quantitation by LC‐MS: Battling the deleterious effects of analyte adsorption

Murphy

Joy

Ouellette

et al. 2020

Analytical Science Advances

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Peptide and protein quantitation by liquid chromatography‐mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low‐bind microcentrifuge tubes and LC vials on the response of a 27‐mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low‐bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low‐bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low‐bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low‐level detection of molecules by mass spectrometry.

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Characterization of Sample Loss Caused by Competitive Adsorption of Proteins in Vials Using Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Cited by 9 publications

References 66 publications

Adsorbed protein film on pump surfaces leads to particle formation during fill‐finish manufacturing

Adsorbed protein film on pump surfaces leads to particle formation during fill‐finish manufacturing

Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry

Improved intact peptide and protein quantitation by LC‐MS: Battling the deleterious effects of analyte adsorption

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