A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies.

Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross-linking, followed by incubation with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP-mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme-linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety.

Improved detection of host cell proteins (HCPs) in a mammalian cell-derived antibody drug using liquid chromatography/mass spectrometry in conjunction with an HCP-enrichment strategy.

RATIONALE: Host cell proteins (HCPs), which are process-related impurities typically present at low levels in recombinant biopharmaceutical products, are often measured using an immunological technique, such as an enzyme-linked immunosorbent assay (ELISA). In contrast to ELISA which only provides the total amount of HCP, liquid chromatography/mass spectrometry (LC/MS) can provide both qualitative and quantitative information about the major HCP species. In this study, an HCP-enrichment step was optimized and combined with LC/MS to identify and determine the relative abundance of HCPs present in a monoclonal antibody (mAb) drug product. METHODS: An NS0 (mouse myeloma) cell-derived mAb drug product, whose total HCP level was less than 100 ng/mg of protein, was subjected to analysis by LC/MS. One-dimensional and two-dimensional chromatography options, together with the off-line HCP enrichment strategy based on Protein A chromatography, were evaluated for optimal HCP detection. RESULTS: With this approach, nineteen HCPs were detected from a therapeutic mAb, an improvement over the detection of only one HCP without depletion. CONCLUSIONS: Compared with other published HCP studies with LC/MS, the HCP-enrichment step in our method enables a more practical and relevant application to approved protein therapeutics, which are mostly mammalian cell-derived products with HCPs present at very low levels.

Polymerase I and transcript release factor regulates lipolysis via a phosphorylation-dependent mechanism.

OBJECTIVE: Polymerase I and transcript release factor (PTRF) is a protein highly expressed in adipose tissue and is an integral structural component of caveolae. Here, we report on a novel role of PTRF in lipid mobilization. RESEARCH DESIGN AND METHODS: PTRF expression was examined in different adipose depots of mice during fasting, refeeding, and after administration of catecholamines and insulin. Involvement of PTRF during lipolysis was studied upon PTRF knockdown and overexpression and mutation of PTRF phosphorylation sites in 3T3-L1 adipocytes. RESULTS: PTRF expression in mouse white adipose tissue (WAT) is regulated by nutritional status, increasing during fasting and decreasing to baseline after refeeding. Expression of PTRF also is hormonally regulated because treatment of mice with insulin leads to a decrease in expression, whereas isoproterenol increases expression in WAT. Manipulation of PTRF levels revealed a role of PTRF in lipolysis. Lentiviral-mediated knockdown of PTRF resulted in a marked attenuation of glycerol release in response to isoproterenol. Conversely, overexpressing PTRF enhanced isoproterenol-stimulated glycerol release. Mass-spectrometric analysis revealed that PTRF is phosphorylated at multiple sites in WAT. Mutation of serine 42, threonine 304, or serine 368 to alanine reduced isoproterenol-stimulated glycerol release in 3T3-L1 adipocytes. CONCLUSIONS: Our study is the first direct demonstration for a novel adipose tissue-specific function of PTRF as a mediator of lipolysis and also shows that phosphorylation of PTRF is required for efficient fat mobilization.

Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes.

The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V(MAX) and K(M) for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1.

A new role for caveolae as metabolic platforms.

The plasma membrane of cells functions as a barrier to the environment. Caveolae are minute invaginations of the membrane that selectively carry out the exchange of information and materials with the environment, by functioning as organizers of signal transduction and through endocytosis. Recent findings of uptake of different metabolites and of lipid metabolism occurring in caveolae, point to a new general function of caveolae. As gateways for the uptake of nutrients across the plasma membrane, and as platforms for the metabolic conversion of nutrients, especially in adipocytes, caveolae are now emerging as active centers for many aspects of intermediary metabolism, with implications for our understanding of obesity, diabetes and other metabolic disorders.

Association and insulin regulated translocation of hormone-sensitive lipase with PTRF.

Polymerase I and transcript release factor (PTRF) is in human adipocytes mainly localized at the plasma membrane. This localization was under control of insulin, which translocated PTRF to the cytosol and nucleus, indicating a novel role for PTRF in insulin transcriptional control. In the plasma membrane PTRF was specifically bound to a triacylglycerol-metabolizing subclass of caveolae containing hormone-sensitive lipase (HSL). In response to insulin PTRF was translocated to the cytosol in parallel with HSL. PTRF and HSL were quantitatively immunoprecipitated from the cytosol by antibodies against either PTRF or HSL. The findings indicate also a novel extranuclear function for PTRF in the control of lipolysis.

Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes.

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

N-terminal processing and modifications of caveolin-1 in caveolae from human adipocytes.

Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.