Kirk J. Leister

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous‐based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for “in‐house” sample handling, tracking, and quality control. Biotechnol. Bioeng. 2010;107: 290–301.

Epidermal Growth Factor and tumor promoters prevent DNA fragmentation by different mechanisms

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

Modeling kinetics of a large‐scale fed‐batch CHO cell culture by Markov chain Monte Carlo method

Markov chain Monte Carlo (MCMC) method was applied to model kinetics of a fed‐batch Chinese hamster ovary cell culture process in 5,000‐L bioreactors. The kinetic model consists of six differential equations, which describe dynamics of viable cell density and concentrations of glucose, glutamine, ammonia, lactate, and the antibody fusion protein B1 (B1). The kinetic model has 18 parameters, six of which were calculated from the cell culture data, whereas the other 12 were estimated from a training data set that comprised of seven cell culture runs using a MCMC method. The model was confirmed in two validation data sets that represented a perturbation of the cell culture condition. The agreement between the predicted and measured values of both validation data sets may indicate high reliability of the model estimates. The kinetic model uniquely incorporated the ammonia removal and the exponential function of B1 protein concentration. The model indicated that ammonia and lactate play critical roles in cell growth and that low concentrations of glucose (0.17 mM) and glutamine (0.09 mM) in the cell culture medium may help reduce ammonia and lactate production. The model demonstrated that 83% of the glucose consumed was used for cell maintenance during the late phase of the cell cultures, whereas the maintenance coefficient for glutamine was negligible. Finally, the kinetic model suggests that it is critical for B1 production to sustain a high number of viable cells. The MCMC methodology may be a useful tool for modeling kinetics of a fed‐batch mammalian cell culture process.

Performance monitoring of a mammalian cell based bioprocess using Raman spectroscopy

Being able to predict the final product yield at all stages in long-running, industrial, mammalian cell culture processes is vital for both operational efficiency, process consistency, and the implementation of quality by design (QbD) practices. Here we used Raman spectroscopy to monitor (in terms of glycoprotein yield prediction) a fed-batch fermentation from start to finish. Raman data were collected from 12 different time points in a Chinese hamster ovary (CHO) based manufacturing process and across 37 separate production runs. The samples comprised of clarified bioprocess broths extracted from the CHO cell based process with varying amounts of fresh and spent cell culture media. Competitive adaptive reweighted sampling (CoAdReS) and ant colony optimization (ACO) variable selection methods were used to enhance the predictive ability of the chemometric models by removing unnecessary spectral information. Using CoAdReS accurate prediction models (relative error of predictions between 2.1% and 3.3%) were built for the final glycoprotein yield at every stage of the bioprocess from small scale up to the final 5000 L bioreactor. This result reinforces our previous studies which indicate that media quality is one of the most significant factors determining the efficiency of industrial CHO-cell processes. This Raman based approach could thus be used to manage production in terms of selecting which small scale batches are progressed to large-scale manufacture, thus improving process efficiency significantly.

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography–mass spectrometry peptide mapping

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (∼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.

Fluorescence Excitation—Emission Matrix (EEM) Spectroscopy for Rapid Identification and Quality Evaluation of Cell Culture Media Components

The application of fluorescence excitation–emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

Comprehensive, quantitative bioprocess productivity monitoring using fluorescence EEM spectroscopy and chemometrics

This study demonstrates the application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative predictive analysis of recombinant glycoprotein production cultured in a Chinese hamster ovary (CHO) cell fed-batch process. The method relies on the fact that EEM spectra of complex solutions are very sensitive to compositional change. As the cultivation progressed, changes in the emission properties of various key fluorophores (e.g., tyrosine, tryptophan, and the glycoprotein product) showed significant differences, and this was used to follow culture progress via multiple curve resolution alternating least squares (MCR-ALS). MCR-ALS clearly showed the increase in the unique dityrosine emission from the product glycoprotein as the process progressed, thus provided a qualitative tool for process monitoring. For the quantitative predictive modelling of process performance, the EEM data was first subjected to variable selection and then using the most informative variables, partial least-squares (PLS) regression was implemented for glycoprotein yield prediction. Accurate predictions with relative errors of between 2.3 and 4.6% were obtained for samples extracted from the 100 to 5000 L scale bioreactors. This study shows that the combination of EEM spectroscopy and chemometric methods of evaluation provides a convenient method for monitoring at-line or off-line the productivity of industrial fed-batch mammalian cell culture processes from the small to large scale. This method has applicability to the advancement of process consistency, early problem detection, and quality-by-design (QbD) practices.

Perturbation of EGF-activated MEK1 and PKB signal pathways by TGF-?1 correlates with perturbation of EGF-induced cyclin D1 and DNA synthesis by TGF-?1 in C3H 10T1/2 cells

In mouse C3H 10T1/2 cells, we previously reported that TGF‐β1 first delays and later potentiates EGF‐induced DNA synthesis corresponding to an inhibition of EGF‐induced cyclin D1 expression at t = 13 h. We report here that in accord with DNA synthesis kinetics, TGF‐β1 initially suppresses EGF‐induced cyclin D1 expression then later releases the inhibition. Furthermore, TGF‐β1 also first decreases and later potentiates the levels of EGF‐activated MEK1/MAPK and PKB, indicating the existence of cross talk between TGF‐β 1– and EGF‐activated signal transduction pathways. PD98059, the specific inhibitor of MEK1, significantly blocks EGF‐induced DNA synthesis, whereas wortmannin, the PI3K inhibitor, exerts a modest inhibitory effect, which suggests that the activation of MEK1‐MAPK pathway plays a major role in EGF‐induced DNA synthesis and the activation of PI3K‐PKB pathway plays a minor role. Upon examination of mechanisms underlying the cross talk, it was discovered that application of TGF‐β1 triggers a rapid association between Raf‐1 and catalytic subunits of PKA, which are reported to be able to inactivate Raf‐1 upon activation. Therefore, TGF‐β1 may activate PKA to inhibit the EGF‐activated MEK1‐MAPK pathway. The wortmannin‐sensitive phosphorylation at the thr389 site is necessary for activation of p70s6K, an important kinase involved in mitogen‐stimulated protein synthesis. Although we found that EGF‐stimulated p70s6K phosphorylates through a MAPK‐dependent and a MAPK‐independent (wortmannin‐sensitive) pathway, TGF‐β1 failed to block EGF‐triggered phosphorylation of p70s6K at thr389 and thr421/ser424 sites, implying that PKB inhibition by TGF‐β1 may result from inhibition of PDK1 activity instead of inhibition of PI3K activity. These data also suggest that TGF‐β1 may selectively perturb certain EGF‐activated MAPK pools. J. Cell. Physiol. 185:107–116, 2000.

Disaggregation of High-Molecular Weight Species During Downstream Processing to Recover Functional Monomer

The use of chaotropic agents to recover functional monomeric material was investigated for the downstream purification of an Fc‐fusion protein containing high levels of high‐molecular weight (HMW) species. In batch studies, chaotropic agents irreversibly disaggregated a majority of the aggregated protein. An integrated processing mode, termed as on‐column disaggregation, was developed in which the protein was captured on Protein A chromatography and then a chaotropic agent was used to simultaneously elute the bound protein and disaggregate the HMW species. On‐column disaggregation process resulted in protein recoveries of >95% and aggregation reduction of ∼50%. Analytical results are presented showing that the recovered monomeric material was comparable to the reference protein in biochemical, biophysical, and pharmacokinetic properties. The kinetic and molecular mechanisms governing protein aggregation and disaggregation will also be elucidated. For the Fc‐fusion protein studied here, incorporation of the disaggregation strategy in both batch and on‐column modes led to an increase of >10% in overall downstream yield.

Cross Validation of Liquid Chromatography–Mass Spectrometry and Lectin Array for Monitoring Glycosylation in Fed-Batch Glycoprotein Production

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC–MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose β1-, terminal N-acetylglucosamine β1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose β1-, while N-acetylglucosamine β1- was less affected. LC–MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC–MS correlated with the detection of the same epitope by the lectin array.