A high-resolution capillary isoelectric focusing method for the determination of therapeutic recombinant monoclonal antibody.

Capillary isoelectric focusing (CIEF) is a common choice for separation and analysis of the charge variants and impurities of therapeutic proteins. In this study, we developed a sensitive CIEF analysis method for determining the charge heterogeneity of therapeutic monoclonal antibody (mAb) using Beckman PA800 plus platform. The mixture of 5% Pharmalyte 8-10.5 and 1% Pharmalyte 3-10 was used to overcome the limitation of using single Pharmalyte 3-10 in detecting charge heterogeneity of basic mAb. This approach largely improved the resolution of the heterogeneous peaks. In addition, 3 M urea and 50 mM arginine (Arg) were used to improve the separation as solubilizer and cathodic stabilizer, respectively. Under optimized condition, both acidic and basic peaks of the mAb were separated well. Method qualification results showed good specificity, precision, and linearity within the concentration range of 0.03-0.20 mg/mL for mAb R1. The method was then used for C-terminal lysine (Lys) variants characterization and glycosylation profiles analysis. Furthermore, it also had a wide application in the clone screening process. The highly sensitive and repeatable results highlighted the wide application prospects of this method in biopharmaceutical industry.

Pre-fractionation of rat liver cytosol proteins prior to mass spectrometry-based proteomic analysis using tandem biomimetic affinity chromatography.

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low-copy-number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre-fractionation were digested and then analyzed by LC-MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un-fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un-fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre-fractionation with MS-based proteomic analysis is simple, low-cost, and effective, providing the prospect of broad application in proteomics.

Comparative analysis of depurination catalyzed by ricin A-chain on synthetic 32mer and 25mer oligoribonucleotides mimicking the sarcin/ricin domain of the rat 28S rRNA and E. coli 23S rRNA.

Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using radiolabeled oligoribonucleotides, two kinds of synthetic 5'-FAM fluorescence-labeled oligoribonucleotide substrates were used to mimic the sarcin/ricin domains of rat 28S rRNA and E. coli 23S rRNA (32mer and 25mer, named as Rat FAM-SRD and E. coli FAM-SRD, respectively). Ricin A-chain was able to specifically release adenine from the first adenosine of the GAGA tetraloop and exhibited specific N-glycosidase activity under neutral and weak acidic conditions with both substrates. However, under more acidic conditions, ricin A-chain was able to release purines from other sites on eukaryotic substrates, but it retained specific depurination activity on prokaryotic substrates. At pH 5.0, the Michaelis constant (K(m)) for the reaction with Rat FAM-SRD (4.57+/-0.28microM) corresponded to that with E. coli FAM-SRD (4.64+/-0.26microM). However, the maximum velocity (V(max)) for ricin A-chain with Rat FAM-SRD was 0.5+/-0.024microM/min, which is higher than that with E. coli FAM-SRD (0.32+/-0.011microM/min).

Combined usage of cascade affinity fractionation and LC-MS/MS for the proteomics of adult mouse testis.

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC-MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC-MS/MS, which resulted in high confidence identification of a total of 1378 non-redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un-fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC-MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.

[Metabolic flux analysis of L-Tryptophan biosynthesis].

The metabolic flux balance model of L-Try synthesis by Corynebacterium glutamicum was constructed in this paper. Using this model, the metabolic flux distribution during the middle and late period were determined and the optimal flux distribution were calculated by linear program of MATLAB software. The analysis results indicate that 24.85% metabolic flux entered the HMP pathway and 75.15% entered the EMP cycle. But comparing to the optimal flux distributions, the production of L-Try should be improved from the genetic manipulation and fermentation control through reducing byproduct of amino acid and decreasing the metabolic flux of TCA and EMP.

Staged-probability strategy of processing shotgun proteomic data to discover more functionally important proteins.

Biologically important proteins related to membrane receptors, signal transduction, regulation, transcription, and translation are usually low in abundance and identified with low probability in mass spectroscopy (MS)-based analyses. Most valuable proteomics information on them were hitherto discarded due to the application of excessively strict data filtering for accurate identification. In this study, we present a staged-probability strategy for assessing proteomic data for potential functionally important protein clues. MS-based protein identifications from the second (L2) and third (L3) layers of the cascade affinity fractionation using the Trans-Proteomic Pipeline software were classified into three probability stages as 1.00-0.95, 0.95-0.50, and 0.50-0.20 according to their distinctive identification correctness rates (i.e. 100%-95%, 95%-50%, and 50%-20%, respectively). We found large data volumes and more functionally important proteins located at the previously unacceptable lower probability stages of 0.95-0.50 and 0.50-0.20 with acceptable correctness rate. More importantly, low probability proteins in L2 were verified to exist in L3. Together with some MS spectrogram examples, comparisons of protein identifications of L2 and L3 demonstrated that the staged-probability strategy could more adequately present both quantity and quality of proteomic information, especially for researches involving biomarker discovery and novel therapeutic target screening.

Characterization and comparison of commercially available TNF receptor 2-Fc fusion protein products.

Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes including peptide mapping, intact mass, charge variant, purity, glycosylation and bioactivity. In contrast, the intact mass and MS/MS analysis of biosimilar 2 revealed a mass difference indicative of a two amino acid residue variance in the heavy chain (Fc) sequences. Comprehensive glycosylation profiling confirmed that biosimilar 2 has significantly low sialylated N-oligosaccharides. Biosimilar 2 also displayed significant differences in charge attributes compared with the reference product. Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties. For a biosimilar development program, comparative analytical data can influence decisions about the type and amount of animal and clinical data needed to demonstrate biosimilarity. Because of the limited clinical experience with biosimilars at the time of their approval, a thorough knowledge surrounding biosimilars and a case-by-case approach are needed to ensure the appropriate use of these products.

A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography.

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC-MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, pI, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.