Application of a Quantitative LC-MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites.

A significant challenge of traditional glycan mapping techniques is that they do not provide site-specific glycosylation information. Therefore, for proteins containing multiple glycosylation sites, the individual glycan species present at a particular site cannot be differentiated from those species present at the other glycosylation sites on the molecule. Quantification of glycoform has previously been demonstrated using a multiattribute method (MAM), which can quantify multiple post-translational modifications including deamidation, oxidation, glycosylation variants, and fragmentation ( Rogers, R. S.; Nightlinger, N. S.; Livingston, B.; Campbell, P.; Bailey, R.; Balland, A. MAbs 2015 , 7 , 881 - 890 ; ref 1). In this paper we describe the application of an MAM based method for site specific quantification of N-linked glycan heterogeneity present on an IgG1 mAb molecule containing two distinct N-linked glycosylation sites: one present on the heavy chain (HC) variable region (Fab) and the other present on the conserved HC constant region (Fc). MAM is a peptide mapping method utilizing mass spectrometry to detect and quantify specific peptides of interest. The ionization properties of the glycopeptides with different classes of glycan structural variants, including high mannose, sialylated, and terminal galactosylated species were studied in detail. Our results demonstrate that MAM quantification of individual glycan species from both the Fab and Fc N-Linked glycosylation sites is consistent with quantification using the traditional hydrophilic interaction liquid chromatography (HILIC) analysis of enzymatically released and fluorescently labeled glycans. Furthermore, no significant impact from the glycoform on the ionization properties of the glycopeptide is observed. Our work demonstrates that the MAM method is a suitable approach for providing quantitative, site-specific glycan information for profiling of N-linked glycans on immunoglobulins.

Capillary zone electrophoresis method for a highly glycosylated and sialylated recombinant protein: development, characterization and application for process development.

A purity method based on capillary zone electrophoresis (CZE) has been developed for the separation of isoforms of a highly glycosylated protein. The separation was found to be driven by the number of sialic acids attached to each isoform. The method has been characterized using orthogonal assays and shown to have excellent specificity, precision and accuracy. We have demonstrated the CZE method is a useful in-process assay to support cell culture and purification development of this glycoprotein. Compared to isoelectric focusing (IEF), the CZE method provides more quantitative results and higher sample throughput with excellent accuracy, qualities that are required for process development. In addition, the CZE method has been applied in the stability testing of purified glycoprotein samples.

Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

Risk-benefit evaluation of on-line high-performance liquid chromatography analysis for pooling decisions in large-scale chromatography.

In the production of a human therapeutic protein from inclusion bodies, product related impurities of very similar size and charge to the product are created as byproducts of the refold process. Their removal is usually challenging even when using chromatography with high performance resins and elution by shallow linear gradients. Additionally, performing this type of separation for commercial production adds increased complexity. To maximize productivity, columns are loaded so high that product elution profiles are not well separated from the impurities and pooling decisions are challenging. In this paper, conventional UV pooling based on fractionation or predefined absorbance based criteria will be compared to pooling based on fast on-line HPLC analytic. The development and implementation in a GMP process will be shown for a specific challenging separation by hydrophobic interaction chromatography. The different approaches have their unique complexities, timelines, uncertainties, and risks during development and implementation as well as during manufacturing. This study presents a probabilistic framework for quantitative comparison of two processes with unequal variability and uncertainty to evaluate the potential benefits of a PAT technology for its routine use in GMP Bioprocess manufacturing.

Large scale demonstration of a process analytical technology application in bioprocessing: use of on-line high performance liquid chromatography for making real time pooling decisions for process chromatography.

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real-time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale-up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale-up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". The application presented here can be extended to other modes of process chromatography and/or HPLC analysis.

Evaluation of pharmacist-managed diabetes mellitus under a collaborative drug therapy agreement.

PURPOSE: The effect of a pharmacist-managed collaborative drug therapy agreement (CDTA) on diabetes mellitus (DM) management in an outpatient setting is evaluated. METHODS: Patients with DM were referred by physicians to the pharmacist for either education or clinical management of DM under the CDTA. A retrospective chart review was conducted between September 2001 and December 2005 and included patients who had laboratory values of interest within one year before and after the initial visit and who had more than two documented visits with the pharmacist. After the pharmacist's intervention in the DM management, glycosylated hemoglobin (HbA(1c)) and low-density lipoprotein cholesterol were compared using a paired sample t test. Average costs for inpatient hospitalization and emergency department (ED) admission were also compared. RESULTS: A total of 110 patients had a mean +/- S.D. of 5.7 +/- 3.9 visits with the pharmacist. A mean reduction in HbA(1c) of 0.7% (p < or = 0.001, n = 93) from 8.9% to 8.2% and a mean reduction in blood glucose of 26.4 mg/dL (p < or = 0.001, n = 99) were achieved. Average costs for inpatient hospitalization and ED admissions were significantly higher in the preintervention period than in the postintervention period for patients with DM as the primary or secondary diagnosis ($2434 versus $636, respectively; p = 0.015). For patients with a primary diagnosis of diabetes, preintervention costs were higher than postintervention costs, but this difference was not significant ($3082 versus $696, respectively; p = 0.100). CONCLUSION: Pharmacist interventions under a CDTA resulted in significant improvements in glucose and HbA(1c) levels in patients with DM. Postintervention costs for inpatient hospitalization and ED services were significantly less than preintervention costs when DM was a primary or secondary diagnosis for the admission.