Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of -4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 ∼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.

Strategic feeding of NS0 and CHO cell cultures to control glycan profiles and immunogenic epitopes of monoclonal antibodies.

The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1-3 Gal that may be immunogenic. We have looked at the effects of a commonly used media supplements of manganese, galactose and uridine (MGU) on Mab production from CHO and NS0 cells in enhancing galactosylation and sialylation as well as the generation of these non-human glycan epitopes. In the absence of the MGU supplement, the humanized IgG1 antibody (Hu1D10) produced from NS0 cells showed a low level of mono- and di-sialylated structures (SI:0.09) of which 75 % of sialic acid was Neu5Gc. The chimeric human-llama Mab (EG2-hFc) produced from CHO cells showed an equally low level of sialylation (SI: 0.12) but the Neu5Gc content of sialic acid was negligible (<3%). Combinations of the MGU supplements added to the production cultures resulted in a substantial increase in the galactosylation of both Mabs (up to GI:0.78 in Hu1D10 and 0.81 in EG2-hFc). However, the effects on sialylation differed between the two Mabs. We observed a slight increase in sialylation of the EG2-hFc Mab by a combination of MG but it appeared that one of the components (uridine) was inhibitory to sialylation. On the other hand, MG or MGU increased sialylation of Hu1D10 substantially (SI:0.72) with an increase that could be attributed predominantly to the formation of Neu5Ac rather than Neu5Gc. The increased level of galactosylation observed with MG or MGU was attributed to an activation of the galactosyl transferase enzymes through enhanced intracellular levels of UDP-Gal and the availability of Mn2+ as an enzymic co-factor. However, this effect not only increased the desirable beta 1-4 Gal linkage to GlcNAc but unfortunately in NS0 cells increased the formation of Galα1-3 Gal which was shown to increase x3 in the presence of combinations of the MGU supplements. Supplementation of media with fetal bovine serum (FBS) increased the availability of free Neu5Ac which resulted in a significant increase in the sialylation of Hu1D10 from NS0 cells. This also resulted in a significant decrease in the proportion of Neu5Gc in the measured sialic acid from the Mab.

Confident Identification of Citrullination and Carbamylation Assisted by Peptide Retention Time Prediction.

The chromatographic behavior of peptides carrying citrulline and homocitrulline residues in proteomic two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS) experiments has been investigated. The primary goal of this study was to determine the chromatographic conditions that allow differentiating between arginine citrullination and deamidation of asparagine based on retention data, improving the confidence of MS-based identifications. Carbamylation was used as a reference point due to a high degree of similarity between modification products and anticipated changes in chromatographic behavior. We applied 2D LC-MS/MS (a high-pH-low-pH reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC)-low-pH RP, and strong cation exchange (SCX)-low-pH RP) to acquire retention data for modified-nonmodified peptide pairs in the four separation modes. Modifications of a standard protein mixture were induced enzymatically (PAD-2) or chemically (urea) for citrullination and carbamylation, respectively. Deamidation occurs spontaneously. Similar retention shifts were observed for all three modifications in a high-pH RP (decrease) and a low-pH RP (increase), thus limiting the applicability of this 2D LC combination. HILIC on bare silica and strong cation exchange separations have been probed to amplify the effect of charge loss upon citrullination, with SCX demonstrating the most differentiating power: the elimination of basic residues upon citrullination/carbamylation results in an ∼58 mM KCl retention decrease, while retention of deamidated products decreases slightly.

Improved therapeutic efficacy of mammalian expressed-recombinant interferon gamma against ovarian cancer cells.

Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HEK293, glycosylated/de-glycosylated) on cytostasis, cell death (MTT, and Guava-ViaCount® flow-cytometry) and apoptotic signalling (Western blot of Cdk2, histone H3, procaspase-3, FADD, cleaved PARP, and caspase-3) was examined. Hydrophilic Interaction Liquid Chromatography determined the structure of N-linked glycans present in HEK293-expressed hIFNγ (hIFNγ-HEK). PEO1 was more sensitive to hIFNγ than SKOV3, but responses were dose-dependent and expression platform/glycosylation status-independent, whereas SKOV3 responded to mammalian-expressed hIFNγ in a dose-independent manner, only. Complex-type oligosaccharides dominated the N-glycosylation pattern of hIFNγ-HEK with some terminal sialylation and core fucosylation. Cleaved PARP and cleaved caspase-3 were not detected in either cell line, but FADD was expressed in SKOV3 with levels increased following treatment. In conclusion, hIFNγ did not induce apoptosis in either cell line. Mammalian- expressed hIFNγ increased cell death in the drug-resistant SKOV3. The presence of FADD in SKOV3, which may inhibit apoptosis through activation of NF-κB, could serve as a novel therapeutic target.

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids.

Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble ß-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 µM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.

Low glucose depletes glycan precursors, reduces site occupancy and galactosylation of a monoclonal antibody in CHO cell culture.

Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose. Glucose depletion occurred in cultures with an initial glucose ≤5.5 mM and seeded at low density (2.5 × 10(5) cells/mL) or at high cell inoculum (≥2.5 × 10(6) cells/mL) at higher glucose concentration (up to 25 mM). Glucose-depleted cultures produced non-glycosylated Mabs (up to 51%), lower galactosylation index (GI <0.43) and decreased sialylation (by 85%) as measured by mass spectrometry and HPLC. At low glucose a reduced intracellular pool of nucleotides (0.03-0.23 fmoles/cell) was measured as well as a low adenylate energy charge (<0.57). Low glucose also reduced GDP-sugars (by 77%) and UDP-hexosamines (by 90%). The data indicate that under glucose deprivation, low levels of intracellular nucleotides and nucleotide sugars reduced the availability of the immediate precursors of glycosylation. These results are important when applied to the design of fed-batch cultures.

Evaluation of Quenching and Extraction Methods for Nucleotide/Nucleotide Sugar Analysis.

Nucleotide sugars are the donor substrates of glycosyltransferases and their availability is known to have an impact on the glycosylation of recombinant proteins including monoclonal antibodies. In addition, the intracellular concentration levels of these metabolites can provide information about the physiological/energetic state of the cell. Therefore, the ability to qualitatively and quantitatively determine the intracellular nucleotides and nucleotide sugars can give valuable insight into the metabolism associated with the glycosylation processes in cells. However, in order to be able to perform a consistent and reliable time specific analysis of these metabolites during a cell culture the metabolism of the cell needs to be stopped immediately at the point of sampling and an efficient extraction needs to be performed. Once the nucleotides and nucleotide sugars are extracted from the cell sample an efficient HPLC method is needed to separate all or most of the metabolites of interest to allow for their identification and quantification. Here, we describe an optimized method for the analysis of the intracellular nucleotide/nucleotide sugar pool in CHO suspension cells which includes protocols for quenching, extraction and HPLC analysis.