ABSTRACT
Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fcâ¯=â¯Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.
Subject(s)
Benzodiazepines/analysis , Immunoconjugates/analysis , Pyrroles/analysis , Benzodiazepines/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Dimerization , Hydrophobic and Hydrophilic Interactions , Immunoconjugates/chemistry , Pyrroles/chemistryABSTRACT
Four mononuclear [(L-L)2 Ru(tatpp)]2+ and two dinuclear [(L-L)2 Ru(tatpp)Ru(L-L)2 ]4+ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4 phen) or 4,7-diphenyl-1,10-phenanthroline (Ph2 phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pHâ 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC50 ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC50 values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC50 values <15â µm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs.
Subject(s)
Antineoplastic Agents/pharmacology , Cholinesterase Inhibitors/pharmacology , Coordination Complexes/pharmacology , Ruthenium/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Line, Tumor , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/toxicity , Cisplatin/pharmacology , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Coordination Complexes/toxicity , DNA Cleavage/drug effects , Humans , Ligands , Mass Spectrometry , Maximum Tolerated Dose , Mice, Inbred BALB C , Mice, Inbred C57BL , Oxidation-Reduction , Proton Magnetic Resonance SpectroscopyABSTRACT
The ruthenium (II) polypyridyl complexes (RPC), Δ-[(phen)2Ru(tatpp)]Cl2 (Δ-[3]Cl2) and ΔΔ-[(phen)2Ru(tatpp)Ru(phen)2]Cl4 (ΔΔ-[4]Cl4, are a new generation of metal-based antitumor agents. These RPCs bind DNA via intercalation of the tatpp ligand, which itself is redox-active and is easily reduced at biologically relevant potentials. We have previously shown that RPC 4(4+) cleaves DNA when reduced by glutathione to a radical species and that this DNA cleavage is potentiated under hypoxic conditions in vitro. Here, we show that 3(2+) also exhibits free radical-mediated DNA cleavage in vitro and that 3(2+) and 4(4+) both exhibit selective cytotoxicity toward cultured malignant cell lines and marked inhibition of tumor growth in vivo. The murine acute toxicity of RPCs 3(2+) and 4(4+) (maximum tolerable doses ~ 65 µmol/kg) is comparable with that for cisplatin (LD50 ~ 57 µmol/kg), but unlike cisplatin, RPCs are generally cleared from the body unchanged via renal excretion without appreciable metabolism or nephrotoxic side effects. RPCs 3(2+) and 4(4+) are shown to suppress growth of human non-small cell lung carcinoma (~83%), show potentiated cytotoxicity in vitro under hypoxic conditions, and induce apoptosis through both intrinsic and extrinsic pathways. The novel hypoxia-enhanced DNA cleavage activity and biologic activity suggest a promising new anticancer pharmacophore based on metal complexes with aromatic ligands that are easily reduced at biologically accessible potentials.
Subject(s)
Antineoplastic Agents/pharmacology , Lung Neoplasms/metabolism , Ruthenium , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation/drug effects , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Coordination Complexes/toxicity , DNA Cleavage/drug effects , Humans , Inhibitory Concentration 50 , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Male , Mice , Mice, Nude , Ruthenium/chemistry , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.
Subject(s)
Chelating Agents/chemistry , Membrane Proteins/isolation & purification , Polyethylene Glycols/chemistry , Polymers/chemistry , Protein Stability/drug effects , Buffers , Membrane Proteins/chemistry , SolubilityABSTRACT
In the absence of dioxygen, the cationic complex [(phen)2Ru(tatpp)Ru(phen)2]4+ (P4+) undergoes in situ reduction by glutathione (GSH) to form a species that induces DNA cleavage. Exposure to air strongly attenuates the cleavage activity, even in the presence of a large excess of reducing agent (e.g., 40 equiv of GSH per P4+), suggesting that the complex may be useful in targeting cells with a low-oxygen microenvironment (hypoxia) for destruction via DNA cleavage. The active species is identified as the doubly reduced, doubly protonated complex H2P4+, and a carbon-based radical species is implicated in the cleavage action. We postulate that the dioxygen concentration regulates the degree to which the carbon radical forms and thus regulates the DNA cleavage activity.