Assessing the long-term stability of anti-drug antibodies in method validation: what is the added value?

The stability of analytes in biological matrices is a critical parameter assessed under bioanalytical method validations. Assessing the stability of anti-drug antibodies (ADA) is, however, not evident given the polyclonal antibody response and the practicality of obtaining reference material from human subjects. Moreover, the same argument that different test articles exhibit different stabilities does not translate to polyclonal antibodies, for which there is a body of literature supporting the stability of human antibodies in undiluted human biological matrix for several years. Herein, the current recommendations from industry leaders and health authorities is summarized, and the literature supporting the stability of ADAs is described to provide a consolidated reference for bioanalytical scientists submitting ADA method validations to regulatory agencies.

Approaching stability challenges for flow cytometry in a regulated bioanalytical environment.

Stability of samples for flow cytometry is a critical parameter since storage period of samples is restricted to only a limited period after collection. For most studies, clinical samples have to be shipped to a testing laboratory, in contrast to preclinical samples, which can be analyzed on-site or off-site. Therefore, evaluating stability is critical to provide flexibility on testing of samples to obtain reliable data. A wide variety of factors contributes to establishing stability from sample collection through acquisition. We provided suggestions for experimental and stability parameters to be taken into consideration when designing a flow cytometry method. The case studies presented represent how certain stability issues were overcome to perform flow cytometry assays in a regulated bioanalytical environment.

Loss of thiol repair systems in human cataractous lenses.

PURPOSE: The purpose of this study was to investigate the thiol repair systems of thioltransferase (TTase) and thioredoxin (Trx) and oxidation-damaged proteins in human cataractous lenses. METHODS: Cataractous lenses in humans (57-85 years of age) were classified into cortical, nuclear, mixed, mature, and hypermature cataract types by using a lens opacity classification system, and were obtained by extracapsular cataract extraction (ECCE) procedure. Cortical and nuclear cataracts were grouped by decreasing order of visual acuity into optical chart reading (R), counting fingers (CF), hand motion (HM), and light perception (LP). ECCE lens homogenate was analyzed for glutathione (GSH) level and enzyme activities of TTase, glutathione reductase (GR), Trx, and thioredoxin reductase (TR). Cortical and nuclear cataractous lenses (8 of each) with visual acuity better than HM were each dissected into cortical and nuclear portions for measurement of glyceraldehyde 3-phosphate dehydrogenase (G3PD) activity. Clear lenses (in humans 49-71 years of age) were used as control. RESULTS: Compared with control, all cataractous lenses lost more than 80% GSH and 70% GR; TR and Trx activity; and 40% to 70% TTase activity, corroborated with the loss in visual acuity. Among cataracts with R and CF visual acuity, cortical cataract lost more cortical G3PD activity (18% of control) than that of nuclear cataract (50% of control), whereas GSH depletion and TTase inactivation were similar in both cataracts. CONCLUSIONS: Thiol repair systems were damaged in all types of cataracts. Cortical and nuclear cataracts showed differential G3PD inactivation in the cortex, implying those 2 type of cataracts might be formed through different mechanisms.

Overexpression of thioredoxin-binding protein 2 increases oxidation sensitivity and apoptosis in human lens epithelial cells.

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200µM, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200µM, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx-ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.

Osmotic stress, not aldose reductase activity, directly induces growth factors and MAPK signaling changes during sugar cataract formation.

In sugar cataract formation in rats, aldose reductase (AR) activity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose/galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-ß, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osmotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-ß and the altered cytotoxic signaling that is observed during sugar cataract formation.

Regulation of cytosolic phospholipase A2 (cPLA2alpha) and its association with cell proliferation in human lens epithelial cells.

PURPOSE: To investigate the molecular mechanism for cytosolic phospholipase A2 (cPLA(2)α) regulation and its association to platelet-derived growth factor (PDGF)-induced cell proliferation. METHODS: cPLA(2)α was examined using human lens epithelial (HLE) B3 cells. Reactive oxygen species (ROS) generation induced by PDGF was analyzed by luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA(2)α gene was cloned via RT-PCR followed by site-directed mutagenesis to construct HLE B3 cells expressing either inactive cPLA(2)α enzyme with S228A mutation (S228A), or cPLA(2)α truncated at the calcium-binding C2 domain (C2D). Activity of cPLA(2)α was measured by arachidonic acid (AA) release from cell membranes using [(3)H]-arachidonic acid prelabeled cells. The effect of intracellular calcium level on cPLA(2)α function was examined by treating cells with ionomycin (calcium influx), thapsgargin (endoplasmic reticulum [ER] calcium store release) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA; calcium chelator). Activation of extracellular signal-regulated kinases (ERK), JNK, p38, or Akt was detected by Western blot analysis using specific antibodies. RESULTS: S228A mutant showed suppressed PDGF-induced reactive oxygen species generation, ERK and JNK activation (no effect on p38 or Akt), and cell proliferation in comparison with the vector alone (Vec) control. Calcium-binding C2 domain cells lost the ability of membrane translocation and activation of cPLA(2)α. PDGF cell signaling was calcium-dependent, and the calcium was supplied either from the external flux or endoplasmic reticulum store. However, enrichment of cellular calcium not only augmented PDGF function, but also demonstrated a cPLA(2)α-dependent calcium-signaling cascade that led to cell proliferation. CONCLUSIONS: cPLA(2)α is regulated by calcium mobilization and mitogen-activated protein kinases (MAPK) activation. Both PDGF mitogenic action and calcium signaling are cPLA(2)α-dependent.

Effect of age on the thioltransferase (glutaredoxin) and thioredoxin systems in the human lens.

PURPOSE: To investigate the effect of age on the key oxidation repair enzymes of the thioltransferase (TTase) and thioredoxin (TRx) systems in the human lens. METHODS: Twenty-three normal human lenses (donor ages, 19-77 years) were grouped into second, third, fifth, sixth, and seventh decades and analyzed for TTase, TRx, glutathione reductase (GR), thioredoxin reductase (TR), and glyceraldehyde-3-phosphate dehydrogenase (G3PD) activities, as well as the glutathione (GSH) pool. Additionally, 19 contralateral lenses of the donor eyes were each divided into cortex and nucleus for enzyme distribution studies. RESULTS: All the enzymes showed similar activity in the cortex and nucleus, regardless of age, but were inactivated to various extents in the older lenses. In the TTase system, both TTase and GR showed activity loss over the five decades, with 70% remaining in the seventh decade, whereas the GSH pool was depleted extensively, with only 35% left in the older lenses. In the TRx system, TRx activity was not affected as much as TR for which only 70% of the activity was found in the seventh decade compared with the second to third decades. Overall, G3PD was more sensitive to age because only 50% activity remained after the sixth decade. CONCLUSIONS: With increasing age there is a gradual activity loss in both the TTase and the TRx systems and a lowered GSH pool. These alterations, compounded with the age-related loss in G3PD activity, may lead to redox and energy imbalance, likely contributing to a higher risk to cataract formation in the aging population.

Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria.

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I.

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing.

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.

Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.

PURPOSE: To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. METHODS: Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. RESULTS: Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. CONCLUSIONS: These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.

Revival of glutathione reductase in human cataractous and clear lens extracts by thioredoxin and thioredoxin reductase, in conjunction with alpha-crystallin or thioltransferase.

Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions. Thioltransferase (TTase, or glutaredoxin) can maintain the homeostasis of lens protein thiols thus protecting against oxidative stress. We investigated whether the Trx/TrxR system can revive GR activity in both the cortex and nucleus of human cataract and clear aged lenses and whether alpha-crystallin and TTase can help this effect. The GR activity in the cortex and nucleus of the cataractous lenses was significantly lower than that of the aged clear lenses. The highest activity in the cortex was observed in the clear aged lenses. The combination of Trx and TrxR revived the activity of GR from both the cortex and nucleus of aged clear lenses. However, in cataract lenses (grade II and grade IV), there was a statistically significant recovery of GR activity in the cortex, but not in the nucleus. No recovery was observed when Trx or TrxR were used separately. Alpha-crystallin successfully revived GR activity in the cortex of cataract grade II lenses, but not in the nucleus. The combination of alpha-crystallin and Trx/TrxR gave a further increase of activity. TTase alone revived some of the GR activity but together with the Trx/TrxR system gave no statistically significant enhancement of GR activity. These results indicate that both disulfide bond formation and protein unfolding are responsible for GR inactivation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) and its possible physiological functions of redox signaling in the eye lens.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) was cloned from human lens epithelial B3 cells (HLE B3) and the recombinant enzyme was purified to homogeneity. The pure enzyme reacted positively with anti-LMW-PTP antibody, displayed tyrosine-specific phosphatase activity and was extremely sensitive to H(2)O(2). The inactivated LMW-PTP could be regenerated by thioltransferase (TTase)/GSH system as demonstrated by both activity assay and by mass spectrometry (MS). The MS study also showed that an intramolecular disulfide bond was formed between C13 and C18 at the active site, and was reduced by the TTase/GSH system. The putative role of LMW-PTP in regulating platelet derived growth factor (PDGF)-stimulated cell signaling was demonstrated in wild type mouse lens epithelial cells (LEC) in which LMW-PTP was transiently inactivated, corroborated with the transient phosphorylation of Tyr857 at the active site of PDGF receptor and the downstream signaling components of Akt and ERK1/2. In contrast, LMW-PTP activity in PDGF-stimulated LEC from TTase(-/-) mice was progressively lost, concomitant with the high basal and sustained high phosphorylation levels at Tyr857, Akt and ERK1/2. We conclude that the reversible LMW-PTP activity regulated by ROS-mediated oxidation and TTase/GSH reduction is the likely mechanism of redox signaling in lens epithelial cells.

The positive feedback role of arachidonic acid in the platelet-derived growth factor-induced signaling in lens epithelial cells.

PURPOSE: Platelet-derived growth factor (PDGF)-stimulated cell proliferation has been associated with reactive oxygen species (ROS)-mediated redox signaling. This study examined the role of arachidonic acid (AA) in PDGF-stimulated ROS generation in human lens epithelial B3 cells (HLE B3). METHODS: PDGF (1 ng/ml)-stimulated ROS generation was examined using dichlorofluorescein (DCFH)-activated fluorescence by laser confocal microscopy while AA (30-150 muM)-stimulated superoxide anion production was measured using lucigenin-amplified chemiluminescence in serum-starved HLE B3 cells. PDGF-stimulated AA release was quantified by cells prelabeled with (3)H-AA with and without the presence of cytosolic phospholipase A(2) (cPLA(2)) inhibitor (AACOCF(3)) and mitogen-activated protein (MAP) kinases (MEK) inhibitor (U0126). Western blot analysis was used to characterize the activated MAP kinase components in cell lysates or protein kinase C (PKC) translocation in isolated cytosolic and membrane fractions. Specific inhibitors to various enzymes were used in the study, including GF109203X for pan protein kinase C (PKC), AACOCF3 for cytosolic phospholipase A2 (cPLA(2)), U0126 for MEK, and DPI for NADPH oxidase. Inhibitors for AA metabolism were also used to examine the role of AA in PDGF-stimulated ROS generation, including CDC and NDGA for pan lipoxygenase, AA861 for 5-lipoxygenase, indomethacin for cycloxygenase, and ketoconazole for cytochrome p450. RESULTS: We found that PDGF-stimulated ROS was eradicated by inhibitors to MEK, cPLA(2), 5-lipoxygenase, NADPH oxidase, or PKC. PDGF-stimulated AA release depended on both active cPLA(2) and ERK1/2. Exogenous AA showed a concentration-dependent ROS generation via NADPH oxidase activation that was insensitive to MEK inhibitor, but sensitive to PKC inhibitor, and could be attenuated by superoxide dismutase (SOD), mannitol, or DPI. This effect of AA was specific as other long chain fatty acids (leinoleic acid, stearic acid), or AA derivatives (eicosa-11Z, 14Z, 17Z-trienoic acid (20:3) and eicosa-11Z, 14Z-dienoic acid (20:2)) were ineffective. Inhibitor to lipoxygenase, in particular the 5-isoform, but not cycloxygenase or cytochrome p450, could diminish AA-stimulated luminescence generation. Western blot analysis showed that AA-treated cells transiently activated ERK1/2 and JNK, but not p38, in a time- and dose-dependent manner that was similar to that of PDGF. Finally, PDGF-stimulated PKC translocation depended on AA release while AA-stimulated PKC translocation was eradicated by lipoxygenase inhibition. CONCLUSIONS: We conclude that PDGF signaling in HLE B3 cells is mediated by AA and its lipoxygenase metabolites, which provide a positive feedback loop for PDGF action, as AA and its metabolites can mobilize PKC and other factors needed for NADPH oxidase assembly and activation for ROS generation to facilitate cell proliferation. We further propose the role of AA in PDGF signaling.

Platelet derived growth factor (PDGF)-induced reactive oxygen species in the lens epithelial cells: the redox signaling.

Low level of reactive oxygen species (ROS) has been shown to play an important role in host defense and mediating mitogen-stimulated cell signaling in several cell types. This study is to identify the mitogen-induced endogenous ROS generation and the range of exogenous H(2)O(2) that initiate redox signaling and cell proliferation in human lens epithelial cells (HLE B3), using platelet-derived growth factor (PDGF) as a model. To detect ROS generation, serum starved HLE cells (1.6 million) were loaded with fluorescent dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), before exposing to PDGF (1 ng ml(-1)). The fluorescence generated from the oxidant-sensitive DCFH, the intracellular product of DCFH-DA hydrolysate, was immediately measured in live cells by confocal laser light microscopy (lambda(Ex)=488 nm, lambda(Em)=522 nm, laser power=10%). PDGF-stimulated cells showed strong transient fluorescence during the 60 min while no fluorescence could be seen in the unstimulated cells. The PDGF-induced fluorescence could be suppressed with cells preloaded with N-acetyl-L-cysteine (NAC, 30 mm), catalase (1 mg ml(-1)), or D-mannitol (100mm). The ability of catalase to penetrate and function in HLE cells was confirmed by western blot, enzyme activity and immunofluorescence microscopic analyses. PDGF induced DNA synthesis within one hour as measured by (3)H-thymidine incorporation, and transiently activated the mitogen-activated protein kinases (MAPKs) of ERK1/2 and JNK. PDGF-stimulated DNA synthesis and MAPK activation were eliminated in the presence of catalase or mannitol. Low levels of H(2)O(2) (10-20 microm) mimicked PDGF in both MAPK stimulation and cell proliferation. In conclusion, the mitogenic stimulus function of PDGF in HLE cells appears to be mediated via ROS to activate MAPKs and cell proliferation, which can be mimicked by low levels of H(2)O(2). It is proposed that the physiological function of ROS, the redox signaling, is present in the HLE cells and may play an important role in the development and maintenance of the lens.

The possible physiological function of thioltransferase in cells.

We sought to study the possible physiological function of thioltransferase (TTase) in combating oxidative damage in the lens epithelial cells. The cells transfected with either TTase-containing plasmid or vector only were compared for their resistance to oxidative stress in the presence of a bolus of H2O2 (0.1 mM) for 3 h. Cells depleted of TTase activity upon cadmium treatment were also examined for the resistance to oxidative stress under the same conditions. TTase activity assay, Western blot, and Northern blot analyses confirmed that hTTase gene was successfully transfected into the HLE B3 cells and was overexpressed. The TTase-transfected cells detoxified H2O2 as efficiently as the control cells but displayed a faster and more complete recovery of oxidatively inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase (GPx) activities and suppressed protein thiolation (PSSG formation). With TTase activity being inhibited by cadmium, the spontaneous reactivation of GAPDH under bolus H2O2 treatment was not accomplished in cadmium-pretreated cells. These data indicate a new physiological function of TTase, which involves in the reactivation of the oxidatively inactivated enzymes through dethiolation; thus this redox-regulating enzyme can protect the human lens epithelial cells and maybe other cell types by preventing them from permanent oxidative damage.