Date of Award

Spring 4-28-2020

Degree Type


Degree Name

PhD. Chemistry


Chemistry and Biochemistry


Yuri Kazakevich, Ph.D.

Committee Member

Mark S Bolgar, Ph.D.

Committee Member

Nicholas Snow, Ph.D.

Committee Member

David Sabatino, Ph.D.

Committee Member

Cecilia Marzabadi, Ph.D.


Poly (alkyl aspartamide) stationary phase; Hydrophobic interaction chromatography; Hybrid mode; Monoclonal antibodies, Ammonium acetate; Linear flow velocity; Gradient steepness; Chromatography


Chromatographic Behavior of Therapeutic Monoclonal Antibodies in Hybrid Hydrophobic Interaction Chromatography (HHIC) using Poly (alkyl aspartamide) Silica Columns

A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging chromatographic technique for the separation of biomolecules under non-denaturing conditions. This technique separates biomolecules in their native form where the difference in surface hydrophobicity is maximized. Hybrid mode of HIC (HHIC) methodology uses HIC stationary phases made with poly (alkyl aspartamide) silica columns, which function as a hybrid form of conventional HIC and reversed-phase chromatography (RPLC). This research provided fundamental knowledge about the impact of chromatographic parameters on the separation of co-formulated mAbs. The influence of mobile phase parameters such as salt concentration, pH and the role of organic modifier and as well as stationary phase parameters on the separation were evaluated. This research demonstrated that an adequate chromatographic separation of mAbs and related biomolecules was achieved using a low ammonium acetate concentration which was not achieved with model proteins previously. The study showed the analytical utility of low ammonium acetate condition (0.5M) to widen the chromatographic elution window by eluting early eluters faster and late eluters later and also an increase in peak capacity. In addition, low ammonium acetate concentration showed a five-fold increase in ESI intensity, which is an advantage for online MS.

In Chapter 1, a brief review of the theory, principles of the methodology and the details about the poly (alkyl aspartamide) silica columns is provided. In addition, the area of interest and the scope of the research is presented. In Chapter 2, the impact of salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related biomolecules is presented. Data demonstrated that using these columns, with low concentrations of ammonium acetate, a small portion of organic solvent is required to elute biomolecules in a reasonable time frame. The research showed chromatographic separation is achievable under low ammonium acetate conditions and also helps to widen the chromatographic window. Assessment of conformational changes in the presence of moderate organic content using orthogonal methods is also presented. This chapter demonstrates the impact of linear flow velocity and gradient time on chromatographic efficiency using these columns. Chapter 3 presented how pH of the mobile phase can help tune the chromatographic separation along with the impact of temperature on the separation.

In the literature it was clearly discussed that to obtain adequate separation using ammonium acetate, a pentyl alkyl chain is required. This fact indicates that hydrophobicity of the stationary phase has a great influence on retention. In Chapter 4, the effect of alkyl chain length and the impact of hydrophobicity on selectivity along with the interdependencies of mobile phase parameters and the stationary phase is presented. HHIC chromatographic parameters such as organic modifier can induce conformational changes in biomolecules. Intrinsic fluorescence was used to gain basic knowledge on the conformational changes of a biomolecule. Chapter 5 provides the experimental results of conformational changes which were obtained using an offline batch mode of intrinsic fluorescence and provided fundamental knowledge about possible conformational changes in the presence of poly (alkyl aspartamide) silica columns using hybrid HIC mobile phases.

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