Navigation Links
Quality control of antibodies using the,,,2100 bioanalyzer and the Protein 200,,,Plus assay

Antibodies are commonly used in diagnostics, as research tools (for example protein arrays) or as biopharmaceutical therapeutics. In all cases the successful production of antibodies requires precise testing and quality control. The key quality criteria include: protein identification, quantitation and the monitoring of purity and stability. Agilent's 2100 bioanalyzer, the first commercial lab-on-a-chip analysis tool, developed in collaboration with Caliper Technologies Corporation, was used to verify the suitability of this technology for antibody quality control. The system allows sizing and analysis proteins of 5 to 200 kDa, depending on the application. In addition, it determines the relative quantitation based on internal standards for each sample, or absolute quantitation based on user-defined calibration standards. The microfabricated chips with distinct microfluidic channels allows antibody analysis under both reducing and non-reducing conditions in a single run. In this Application Note we demonstrate the use of the system for the evaluation of a stress stability test of two different antibodies.
Experiments and Results
Antibody samples were kindly provided from a customer in the pharmaceutical industry. For the first sample a stress stability test was performed for one month at 40 C, for the second sample the stress test was done for 12 weeks at 40 C. The standard samples and the stress test samples were analyzed under reducing conditions on the Agilent 2100 bioanalyzer using a Protein 200 Plus LabChip kit and the dedicated Protein 200 Plus software assay. All chips were prepared according to the protocol provided with the Protein 200 Plus LabChip kit. The kit includes 25 chips, spin filters and all the reagents needed for the experiments including the Protein 200 Plus ladder as well as the upper and lower marker premixed in the sample buffer.
To validate the performance of the instrument three individual users measured the samples on two separate instruments using two lots of reagents on different days. Thus a total number of twelve chips were run. The samples were diluted 1:3 in PBS buffer (Life Technologies GmbH, Karlsruhe, Germany) in order to adjust the concentration of the samples to approximately 5 mg/ml total protein concentration. Sample preparation was performed individually for each chip. All samples were loaded twice on each chip. Therefore, 24 analyses per sample were evaluated using the following Protein 200 Plus assay settings for peak integration: minimum peak height of 0.1, minimum peak width of 0.1 and slope threshold of 4.0.

An example of the performed analysis is depicted in figure 1. Figure 1A shows the gel-like image of the four different antibody samples (2 standards, 2 test samples) aligned to the ladder sample. The ladder includes proteins of known sizes, was analyzed on each chip and was used for size determination. Figure 1B displays the overlay of the corresponding electropherograms. Both standard samples show almost only two peaks corresponding to the light (LC) and heavy chain (HC) of the antibody respectively. After incubation of the antibodies at elevated temperatures additional peaks appear in the electropherograms (marked with an arrow) due to degradation of the antibody or the formation of aggregates, larger products. Table 1 shows the results of the validation study. On average 98.76 % of standard antibody sample 1 corresponds to the light and heavy chain of the antibody. After incubation for one month at 40C the two chains represent only 93.78% of the whole protein a mount detected. Antibody 2 was incubated for 12 weeks at 40C and the proportion of light and heavy chain decreases from an average of 98.38 % to 85.34 %. An excellent reproducibility below 2 % CV was achieved

Conclusion
In this study we show that the Agilent 2100 bioanalyzer can be used as an ideal tool to study the stability of antibodies in stress tests with excellent reproducibility. This application is commonly used as a quality control step in QA/QC departments in order to trigger typical degradation and aggregation patterns for a specific antibody. Those patterns will be used in the down stream processes of antibody production and formulations.
Traditional denaturing sodium dodecylsulfate-polyacrylamide gel electrophoreses (SDS-PAGE) still serves as one of the standard methods for the QC of antibodies. SDS-PAGE is labor-intensive, timeconsuming and difficult to standardize (1-3). In contrast the Agilent 2100 bioanalyzer provides a fast, standardized method with automated and detailed data analysis.



'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
2. Generate High-Quality, Directional Plasmid cDNA Libraries
3. Quality assurance of RNA derived from laser microdissected tissue samples obtained by the PALM MicroBeam System using the RNA 6000 Pico LabChip kit.
4. High Quality RNA Isolation Services
5. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
6. Preparation of PCR Quality Template DNA from a Wide Range of Tissues Using InstaGene Matrix
7. Using the Agilent 2100 bioanalyzer for quality control of protein samples prior to MS-analysis
8. Advancing the quality control methodology to assess isolated total RNA and generated fragmented cRNA
9. Evaluation of RNA isolated from human tissues: quality control for surgical and postmortem samples
10. Outstanding transfection rates using the Multiporator
11. Outstanding transfection rates using the Multiporator
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/18/2016)... , ... May 18, 2016 , ... Ryan Benton was ... life expectancy in the late teens to early twenties. DMD is a relatively common ... at the age of 22, Benton’s condition was critical. He met with the founder ...
(Date:5/17/2016)... , ... May 17, 2016 , ... ... and BioSmartSA, a healthcare consultancy based in Saudi Arabia, have formed a partnership ... to healthcare providers in the Kingdom of Saudi Arabia (KSA). , The ...
(Date:5/17/2016)... 17, 2016 Strekin AG, ... Basel, Switzerland announced today the ... P38 mitogen-activated protein kinase.      (Logo: ... will build the necessary research foundation for the ... MAP Kinases play fundamental roles. Pamapimod has a ...
(Date:5/17/2016)... ... 17, 2016 , ... HOLLOWAY AMERICA, a stainless steel pressure vessel and tank ... on Thursday, May 19 in St. Louis, Missouri. The event will be held at ... a vendor showcase during the early afternoon of the event, where the company’s representatives ...
Breaking Biology Technology:
(Date:5/20/2016)... , May 20, 2016  VoiceIt is ... partnership with VoicePass. By working together, ... experience.  Because VoiceIt and VoicePass take slightly different ... engines increases both security and usability. ... excitement about this new partnership. "This ...
(Date:5/9/2016)... Elevay is currently known as ... for high net worth professionals seeking travel for work ... world, there is still no substitute for a face-to-face ... your deal with a firm handshake. This is why ... of citizenship via investment programs like those offered by ...
(Date:4/28/2016)... First quarter 2016:   , Revenues amounted to ... of 2015 The gross margin was 49% (27) ... operating margin was 40% (-13) Earnings per share rose ... was SEK 249.9 M (21.2) , Outlook   ... The operating margin for 2016 is estimated to exceed ...
Breaking Biology News(10 mins):