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Dive into the research topics where Robert L. Dufield is active.

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Featured researches published by Robert L. Dufield.


Biotechnology and Bioengineering | 2010

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Maire H. Caparon; Kevin J. Rust; Alan K. Hunter; Joseph K. McLaughlin; Kristen E. Thomas; John T. Herberg; Robert E. Shell; Paul B. Lanter; Bruce F. Bishop; Robert L. Dufield; Xing Wang; Sa V. Ho

Apolipoprotein A 1 Milano (ApoA‐1M), the protein component of a high‐density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co‐purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA‐1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process. Biotechnol. Bioeng. 2010; 105: 239–249.


Biotechnology Progress | 2009

Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A‐IMilano in an industrial HIC unit operation

Alan K. Hunter; Xing Wang; Eric J. Suda; John T. Herberg; Robert E. Shell; Kristin E. Thomas; Robert L. Dufield; Mark E. Gustafson; Ned M. Mozier; Sa V. Ho

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A‐IMilano (apo A‐IM) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale‐up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co‐immunoprecipitation and Western blotting techniques. Two‐dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory‐scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign.


Journal of Chromatography A | 2010

Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast

Melissa D. Zolodz; John T. Herberg; Halyna E. Narepekha; Emily Raleigh; Matthew R. Farber; Robert L. Dufield; Denis M. Boyle

Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins.


mAbs | 2018

Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics

T. Jennifer Lin; Kathryn M. Beal; Paul W. Brown; Heather S. DeGruttola; Mellisa Ly; Wenge Wang; Chia H. Chu; Robert L. Dufield; Gerald Fries Casperson; James A. Carroll; Olga V. Friese; Bruno Figueroa; Lisa Marzilli; Karin Anderson; Jason C. Rouse

ABSTRACT Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.


Aaps Journal | 2016

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease

Yin Luo; Olga V. Friese; Herbert A. Runnels; Lakshmi Khandke; Gary W. Zlotnick; Ann Aulabaugh; Thomas Gore; Eugene Vidunas; Stephen W. Raso; Elena Novikova; Emilia Byrne; Michael R. Schlittler; Donald Stano; Robert L. Dufield; Sandeep Kumar; Annaliesa S. Anderson; Kathrin U. Jansen; Jason C. Rouse


Chemical Engineering Science | 2009

Reaction kinetics and optimization of the copper-catalyzed oxidation of ApoA-1M

Sa V. Ho; Joseph K. McLaughlin; Kristen E. Thomas; Eric J. Suda; John T. Herberg; Robert L. Dufield; Alan K. Hunter


Archive | 2003

Antibody disulfide isomers, use thereof, and methods of analyzing same

Ned M. Mozier; Robert L. Dufield; Jianming Mo; Gary S. Bild


Archive | 2012

Recombinant apoa-1m from engineered bacteria

Marie H. Caparon; Kevin J. Rust; Alan K. Hunter; Joseph K. McLaughlin; Kristen E. Thomas; John T. Herberg; Robert E. Shell; Paul B. Lanter; Bruce F. Bishop; Robert L. Dufield; Xing Wang; Sa V. Ho


Archive | 2011

Molécules de liaison 4-1bb

Bianca Ahrens; Sangita M. Baxi; Simon Bergqvist; Regis Doyonnas; Robert L. Dufield; Mark William Elliott; Timothy S. Fisher; Richard Michael Jerome; Heather Laurence Jones; Cris Kamperschroer; Kathrin Ladetzki-Baehs; Victoria A. Love; Theodore Oliphant; Adekunle Olatunbosun Onadipe; Wenning Qin; Vinay Radhakrishnan; Allison Rohner; Leslie L. Sharp; Michael Tesar; Kristin E. Thomas; Libbey Anne Yates; Daisy Marie Ziegemeier; Moritz Zulley


Archive | 2011

4-1bb-bindemoleküle

Bianca Ahrens; Sangita M. Baxi; Simon Bergqvist; Regis Doyonnas; Robert L. Dufield; Mark William Elliott; Timothy S. Fisher; Richard Michael Jerome; Heather Laurence Jones; Cris Kamperschroer; Kathrin Ladetzki-Baehs; Victoria A. Love; Theodore Oliphant; Adekunle Olatunbosun Onadipe; Wenning Qin; Vinay Radhakrishnan; Allison Rohner; Leslie L. Sharp; Michael Tesar; Kristin E. Thomas; Libbey Anne Yates; Daisy Marie Ziegemeier; Moritz Zulley

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