Jason A. Starkey

Hybrid Capillary/Microfluidic System for Comprehensive Online Liquid Chromatography-Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry

A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests. Analysis of a complex mixture of peptides yielded a peak capacity of approximately 1400 in 50 min. Three replicate runs demonstrated mean reproducibility for LC retention and CE migration times of 0.32% and 0.75% relative standard deviation (RSD), respectively. The same LC-CE-MS method was also used to characterize the N-linked glycosylation of a monoclonal antibody. Glycopeptides from two different N-linked glycosylation sites were separated from all other tryptic peptides and identified using MS data. The relative amounts of each glycoform and total site occupancy were quantified using LC-CE-MS data.

Assessment of Physical Stability of an Antibody Drug Conjugate by Higher Order Structure Analysis: Impact of Thiol- Maleimide Chemistry

ABSTRACTPurposeTo provide a systematic biophysical approach towards a better understanding of impact of conjugation chemistry on higher order structure and physical stability of an antibody drug conjugate (ADC).MethodsADC was prepared using thiol-maleimide chemistry. Physical stabilities of ADC and its parent IgG1 mAb were compared using calorimetric, spectroscopic and molecular modeling techniques.ResultsADC and mAb respond differently to thermal stress. Both the melting temperatures and heat capacities are substantially lower for the ADC. Spectroscopic experiments show that ADC and mAb have similar secondary and tertiary structures, but these are more easily destabilized by thermal stress on the ADC indicating reduced conformational stability. Molecular modeling calculations suggest a substantial decrease in the conformational energy of the mAb upon conjugation. The local surface around the conjugation sites also becomes more hydrophobic in the ADC, explaining the lower colloidal stability and greater tendency of the ADC to aggregate.ConclusionsComputational and biophysical analyses of an ADC and its parent mAb have provided insights into impact of conjugation on physical stability and pinpointed reasons behind lower structural stability and increased aggregation propensity of the ADC. This knowledge can be used to design appropriate formulations to stabilize the ADC.

Analysis of Identity, Charge Variants, and Disulfide Isomers of Monoclonal Antibodies with Capillary Zone Electrophoresis in an Uncoated Capillary Column

A set of related capillary zone electrophoresis (CZE) methods have been developed for the analysis of identity, charge variants, and disulfide isoforms of IgG monoclonal antibodies (mAbs). These methods utilize an uncoated capillary column. The combined use of concentrated zwitterionic (e-amino-caproic acid) buffer and acid flushing was effective in minimizing the adsorption of protein to the inner wall of a bare capillary. Under these conditions, a selective and reproducible separation of multiple IgG1 and IgG2 monoclonal antibodies (mAbs) was obtained with a long capillary column (40 cm effective length), allowing the reliable identification of different mAbs by migration time. A rapid ( approximately 10 min) and selective separation of charged variants of IgG mAbs was attained using a short capillary column (10 cm effective length). Finally, the addition of urea in the separation buffer resulted in the separation of disulfide isoforms of IgG2 mAbs by CZE. CZE methods using an uncoated capillary column offer a versatile, generic, and economical approach to the evaluation of identity, charge heterogeneity, and disulfide isoforms of IgG antibodies.

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis-Mass Spectrometry.

In this work, we utilize capillary electrophoresis-mass spectrometry (CE-MS) in an integrated microfluidic platform to analyze an intact, lysine-linked antibody drug conjugate (ADC) in order to assess post translational modifications and drug load variants. The initial charge heterogeneity of the unconjugated IgG-2 monoclonal antibody (mAb) was assessed by separating intact charge variants. Three main charge variants were resolved in the CE dimension. These variants were attributed to pyroglutamic acid formation and decarboxylation on the primary structure of the mAb through characteristic mass shifts and changes in electrophoretic mobility. Additionally, glycoforms of the antibody charge variants were identified in the deconvoluted mass spectra. The observed glycoforms and their distribution compared favorably to a released N-glycan analysis performed on the mAb. After conjugation, the ADC was analyzed using the same microchip CE-MS method. The addition of a drug load resulted in a decrease in mobility and an increase in mass of 3145 Da. Five main species that differed in their respective drug-to-antibody ratios (DAR) were fully resolved in the CE separation, with each DAR displaying the same variant population observed on the unconjugated mAb. A DAR range of 0-4 was observed with an average of 1.7 drug loads. The DAR distribution generated from the microfluidic CE-MS data compared favorably to results from infusion-ESI-MS and imaging CE (iCE) analysis of the ADC, techniques commonly used for intact mAb and ADC characterization.

Characterization and Higher-Order Structure Assessment of an Interchain Cysteine-Based ADC: Impact of Drug Loading and Distribution on the Mechanism of Aggregation

The impact of drug loading and distribution on higher order structure and physical stability of an interchain cysteine-based antibody drug conjugate (ADC) has been studied. An IgG1 mAb was conjugated with a cytotoxic auristatin payload following the reduction of interchain disulfides. The 2-D LC-MS analysis shows that there is a preference for certain isomers within the various drug to antibody ratios (DARs). The physical stability of the unconjugated monoclonal antibody, the ADC, and isolated conjugated species with specific DAR, were compared using calorimetric, thermal, chemical denaturation and molecular modeling techniques, as well as techniques to assess hydrophobicity. The DAR was determined to have a significant impact on the biophysical properties and stability of the ADC. The CH2 domain was significantly perturbed in the DAR6 species, which was attributable to quaternary structural changes as assessed by molecular modeling. At accelerated storage temperatures, the DAR6 rapidly forms higher molecular mass species, whereas the DAR2 and the unconjugated mAb were largely stable. Chemical denaturation study indicates that DAR6 may form multimers while DAR2 and DAR4 primarily exist in monomeric forms in solution at ambient conditions. The physical state differences were correlated with a dramatic increase in the hydrophobicity and a reduction in the surface tension of the DAR6 compared to lower DAR species. Molecular modeling of the various DAR species and their conformers demonstrates that the auristatin-based linker payload directly contributes to the hydrophobicity of the ADC molecule. Higher order structural characterization provides insight into the impact of conjugation on the conformational and colloidal factors that determine the physical stability of cysteine-based ADCs, with implications for process and formulation development.

Characterization of Mammalian Cell Culture Raw Materials by Combining Spectroscopy and Chemometrics

Two of the primary issues with characterizing the variability of raw materials used in mammalian cell culture, such as wheat hydrolysate, is that the analyses of these materials can be time consuming, and the results of the analyses are not straightforward to interpret. To solve these issues, spectroscopy can be combined with chemometrics to provide a quick, robust and easy to understand methodology for the characterization of raw materials; which will improve cell culture performance by providing an assessment of the impact that a given raw material will have on final product quality. In this study, four spectroscopic technologies: near infrared spectroscopy, middle infrared spectroscopy, Raman spectroscopy, and fluorescence spectroscopy were used in conjunction with principal component analysis to characterize the variability of wheat hydrolysates, and to provide evidence that the classification of good and bad lots of raw material is possible. Then, the same spectroscopic platforms are combined with partial least squares regressions to quantitatively predict two cell culture critical quality attributes (CQA): integrated viable cell density and IgG titer. The results showed that near infrared (NIR) spectroscopy and fluorescence spectroscopy are capable of characterizing the wheat hydrolysates chemical structure, with NIR performing slightly better; and that they can be used to estimate the raw materials’ impact on the CQAs. These results were justified by demonstrating that of all the components present in the wheat hydrolysates, six amino acids: arginine, glycine, phenylalanine, tyrosine, isoleucine and threonine; and five trace elements: copper, phosphorus, molybdenum, arsenic and aluminum, had a large, statistically significant effect on the CQAs, and that NIR and fluorescence spectroscopy performed the best for characterizing the important amino acids. It was also found that the trace elements of interest were not characterized well by any of the spectral technologies used; however, the trace elements were also shown to have a less significant effect on the CQAs than the amino acids.

Analytical subcloning of a clonal cell line demonstrates cellular heterogeneity that does not impact process consistency or robustness

During development of a cell line intended to support production of an IgG2 monoclonal antibody, a sequence variant caused by a genetic mutation was identified in the bulk drug substance. Gene copy number analysis together with the level of the observed variant in genomic DNA indicated that the master cell bank was a mixed population of cells; some harboring the variant copy and some mutation free. Since the cell bank had been single‐cell cloned, this variant could be used as a biomarker to demonstrate either that the bank was not derived from a single cell, or that the variant was a result of a post‐cloning genetic event, leading to a mixed population of cells. The sequence variant was only present in a small percentage of subclones, confirming the hypothesis that the cell bank was indeed a mixed population. Interrogation of subclones via Southern blot analysis revealed that almost all subclones had very similar transgene integrant structures, suggesting that the cell bank was likely derived from a single cell, and the cellular event that yielded the sequence variant was a post‐cloning event. Further, there were likely several other post‐cloning events that impacted transgene loci, leading to a population of related, yet genetically distinct cells comprising the cell bank. Despite this, the heterogeneous bank performed consistently in a bioprocess across generational age with comparable product quality. These results experimentally demonstrate the heterogeneity of a cell bank derived from a single cell, and its relationship to process consistency.