Greg Conley

The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process

Increased upstream productivity and the continuous pressure to deliver high quality drug product have resulted in the development of new separation technologies and platform strategies for downstream purification processes of monoclonal antibodies (mAb). In this study, the separation attributes of three mixed-mode resins, Mercapto-Ethyl-Pyridine (MEP) hydrophobic charge induction resin, Capto adhere multi-modal anion exchange resin, and ceramic hydroxyapatite/fluoroapatite (CHT/CFT) resins, were investigated to define their roles in monoclonal antibody purification processes. We demonstrated that the multi-modal nature of ligands on mixed-mode resins allows the separation resolution to be honed, either through a single dominant mechanism or through mix-modal balanced purification strategies. In addition, the three mixed-mode resins present different purification powers for different types of impurities. We also demonstrated that besides enhancing chromatography separation and improve product quality, especially for high molecular weight (HMW) aggregate reduction, mixed-mode resins can also help to improve process efficiency in industrial-scale mAb drug manufacturing. Our results underscore the importance of selecting appropriate chromatography resins during DSP design to obtain the best overall process outcome.

Development of Mammalian Serum Albumin Affinity Purification Media by Peptide Phage Display

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide‐constrained cyclic peptide phage‐display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX‐236, also bound well to several mammalian serum albumins (SA). Immobilized DX‐236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX‐236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR–directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1–104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (KD value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor–binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 Å. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I–induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR.

Evaluation of protein engineering and process optimization approaches to enhance antibody drug manufacturability.

A potent single digit picomolar fully human monoclonal antibody (hMAb) inhibitor with a high degree of specificity to the antigen of interest was identified from a phage display library. The hMAb, however, exhibited a high degree of hydrophobicity and easily formed insoluble aggregates when purified using a Protein A based generic process. Strategies were designed using both protein engineering and process development approaches to optimize the molecules amino acid sequence and its behavior in process conditions. The insoluble aggregation issue was brought under control by one single amino acid mutation in CDR region or by switching to non‐ProA based purification process. Our study therefore presents the rational manufacturability design for future monoclonal antibody product and its purification process under the quality by design concept by either engineering the drug molecule to adapt existing platform process or optimizing the process to fit the specific properties of the drug product. Biotechnol. Bioeng. 2011;108: 2634–2644.

Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A.

Abstract Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K m of 33 μM and a k cat of 0.3 s-1 (k cat/K m=9.1×103 M-1 s-1). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK a values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K i values of 1.7±0.2 and 7.4±1.5 nM for the F2 and D9 antibodies, respectively.