Advisor(s)

William S. Hancock

Contributor(s)

Paul Vouros, Zhaohui S. Zhou, Penny J. Beuning

Date of Award

2011

Date Accepted

8-2011

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Science. Department of Chemistry and Chemical Biology.

Keywords

chemistry, analytical chemistry, liquid chromatography - mass spectrometry (LC-MS), Fc-fusion antibody drugs/

Subject Categories

Antibody-drug conjugates, Proteomics, Liquid chromatography

Disciplines

Biochemistry

Abstract

Therapeutic protein drugs produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. Characterization of these protein drugs is very important because it relates to the efficacy and safety issues. In order to dose drug effectively, pharmacokinetics studies that can provide guidance are also critical.

This dissertation focuses on the characterization and pharmacokinetic study of Fc-fusion antibody drugs using liquid chromatography - mass spectrometry (LC-MS) based proteomic technologies. A proteomic study of microvascular endothelium proteins from mouse brain and characterization of anti-Neu5Gc autoantibodies are also presented.

Chapter 1 reviews current methodologies of proteomic analysis in bioanalytical research and the application of proteomic analysis to monoclonal antibody (mAb) therapeutics which includes Fc fusion protein technology and bioanalysis of recombinant proteins and antibodies.

Chapter 2 is a proteomic study of microvascular endothelium proteins from mouse brain. Tissue sample extraction and compatible LC-MS analysis for the proteomic study of mouse brain were developed. The techniques involved the handling of minute amounts of cells (less than 5000 cells) isolated by double label, immuno-laser capture microdissection (LCM) and the use of a linear ion trap (IT) coupled with a Fourier Transform mass spectrometer (LTQ-FT MS) for characterization. Sufficient numbers of proteins with high biological interests were identified.

In chapter 3, different analysis platforms with LC-MS for pharmacokinetic (PK) studies of protein drugs in serum were developed. These analysis platforms can be selectively used for different types of protein drugs that ranged from a very specific for a particular drug (antibody enrichment), to a less specific for any antibody drugs with Fc domain (protein A enrichment), and to a very general method that can be used for any protein drugs (albumin depletion method). High sensitivity (ng/mL) and wide dynamic range (5 orders of magnitude) were achieved for PK studies of the protein drugs. In addition, these analysis platforms can be selectively applied for PK studies of drugs with different requirements of development time and resources. For example, the antibody enrichment method can be used in a high throughput manner but limited to a specific protein drug. On the other hand, the albumin depletion method can be applied to many types of protein drugs, but with the laborious sample preparation steps. When anti-drug antibodies are not available for antibody drugs, or the sensitivity requirement is not stringent, using protein A enrichment (without using SDS-PAGE) seems to be a good choice for PK studies that require high throughput.

Chapter 4 is a characterization of proteolytic degradation reactions observed in the production of a Fc-fusion antibody (GLP-1) which include identification of the product's cleavage sites and responsible proteases in the host Chinese Hamster Ovary (CHO) cells through the study of cell lysate, spent media, and isolates from downstream purification steps. The LC-MS method (nanoLC coupled to LTQ-FTMS) together with the multi-enzyme digestion approach identified the major cleavage site in the fusion antibody as arginine residue 30. In addition, a proteomic analysis on both a lysate of the CHO cells and spent medium were performed to gain information about the protease responsible for this cleavage. Approximately 215 and 250 CHO proteins in the cell lysate and spent medium respectively were identified in this analysis. Among these proteins, the most likely candidate being responsible for the antibody degradation was identified, which is called HtrA serine peptidase 1 (HTRA1), a serine-threonine protease. The methodology described here should provide an effective way to study protease associated losses in the manufacturing processes, as well as facilitate optimization of cellular production yells.

In Chapter 5, human Anti-Neu5Gc autoantibodies isolated from therapeutic preparations of left-overs of human IVIG (IgG) preparations by multi-affinity approaches were characterized. The LC-MS method (nanoLC coupled to LTQ-FTMS) together with our multi-enzyme digestion approach determined the partial sequences in both constant and variable regions. In addition, characteristic peptide sequences were selected and used to calculate the IgG subclass distributions of these anti-Neu5Gc autoantibodies. This methodology should provide a way to characterize these important molecules and help to understand the origins of these antibodies and their significance as biomarkers of cancer.

Document Type

Dissertation

Rights Holder

Qiaozhen Lu


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