In-line fiber optical sensor for detection of IgG aggregates in affinity chromatography.

Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.

Ion exchange chromatography of monoclonal antibodies: effect of resin ligand density on dynamic binding capacity.

Dynamic binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.

Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins.

Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11-20mg/mL), and ligand density (77-138 micromol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200mg/mL was obtained at the optimum pore size and dextran content.

Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.

Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.

A novel and conserved pocket of human kappa-Fab fragments: design, synthesis, and verification of directed affinity ligands.

Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.

Rational design, synthesis, and verification of affinity ligands to a protein surface cleft.

The structure-based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas alpha-amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty-three compounds were selected for synthesis and screened in solution for binding toward alpha-amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand-coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.

Structure-based discovery of a new affinity ligand to pancreatic alpha-amylase.

A ligand useful for affinity capture of porcine pancreatic alpha-amylase was found by virtual screening of the commercially available compound data base MDL Available Chemicals Directory. Hits from the virtual screening were investigated for binding by nuclear magnetic resonance (NMR) and surface plasmon resonance. Selected compounds were tested for inhibition of the enzyme using a NMR-based assay. One of the binders found was covalently coupled to a chromatographic resin and a column, packed with this resin, could retain alpha-amylase, which subsequently was eluted by introduction of the known inhibitor acarbose to the elution buffer.