The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.
The purpose of this work is to determine the cause of the cyclization of the N-terminal glutamine in recombinant proteins and monoclonal antibodies. This cyclization reaction commonly occurs on the N-terminal of light and/or heavy chains of antibodies and leads to heterogeneity of the final product. Two model peptides and an antibody containing an N-terminal glutamine were used to investigate the formation of N-terminal pyro- glutamic acid under various experimental conditions and different stages of the biosynthetic process. LC-MS analysis was used to separate and quantify the N-terminal variants. Experiments prove that the cyclization reaction is spontaneous and highly dependent on temperature and buffer composition and less dependent on pH. The conditions presented in most biopharmaceutical processes accelerate the formation of this variant. The majority of the near complete conversion (>95%) of N-terminal glutamine to pyro-glutamic acid commonly observed for antibodies appears to occur inside the bioreactor with only a small contribution from purification, formulation, and analytical preparation.
The thrombin-binding DNA aptamer was used for affinity capture of thrombin in MALDI-TOF-MS. The aptamer was covalently attached to the surface of a glass slide that served as the MALDI surface. Results show that thrombin is retained at the aptamer-modified surface while nonspecific proteins, such as albumin, are removed by rinsing with buffer. Upon application of the low-pH MALDI matrix, the G-quartet structure of the aptamer unfolds, releasing the captured thrombin. Following TOF-MS analysis, residual matrix and protein can be washed from the surface, and buffer can be applied to refold the aptamers, allowing the surface to be reused. Selective capture of thrombin from mixtures of thrombin and albumin and of thrombin and prothrombin from human plasma was demonstrated. This simple approach to affinity capture, isolation, and detection holds potential for analysis, sensing, purification, and preconcentration of proteins in biological fluids.
Capture and detection of Immunoglobulin E (IgE) in simple solution and in human serum using an aptamer-modified probe surface for affinity Matrix-Assisted Laser Desorption-Ionization Mass Spectroscopy (affinity MALDI-MS) detection is reported. Detectable signals were obtained for 1 amol of IgE applied either in a single, 1 μL application of 1 pM IgE or after 10 successive, 1 μL applications of 100 fM IgE. In both cases, the surface was rinsed after each application of IgE to remove sample concomitants including salts and free or non-specifically associated proteins. Detection of native IgE, which is the least abundant of the serum immunoglobulins and occurs at sub-nM levels, in human serum was demonstrated and interference from the high abundance immunoglobulins and albumin was investigated. The aptamermodified surface showed high selectivity towards immunoglobulins in serum, with no significant interference from serum albumin. Addition of IgE to the serum suppressed the signals from the other immunoglobulins, confirming the expected selectivity of the aptamer surface towards IgE. Dilution of the serum increased the selectivity toward IgE; the protein was detected without interference in a 10,000-fold dilution of the serum, which is consistent with detection of IgE at amol (pM) levels in standard solutions.
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