Small conformational changes in IgG1 detected as acidic charge variants by cation exchange chromatography.

Fully characterizing the post-translational modifications present in charge variants of therapeutic monoclonal antibodies (mAbs), particularly acidic variants, is challenging and remains an open area of investigation. In this study, to test the possibility that chromatographically separated acidic fractions of therapeutic mAbs contain conformational variants, we undertook a mAb refolding approach using as a case study an IgG1 that contains many unidentified acidic peaks with few post-translational modifications, and examined whether different acidic peak fractions could be generated corresponding to these variants. The IgG1 drug substance was denatured by guanidine hydrochloride, without a reducing agent present, and gradually refolded by stepwise dialysis against arginine hydrochloride used as an aggregation suppressor. Each acidic chromatographic peak originally contained in the IgG1 drug substance was markedly increased by this stepwise refolding process, indicating that these acidic variants are conformational variants. However, no conformational changes were detected by small-angle X-ray scattering experiments for the whole IgG1, indicating that the conformational changes are minor. Chromatographic, thermal and fluorescence analyses suggested that the conformational changes are a localized denaturation effect centred around the aromatic amino acid regions. This study provides new insights into the characterization of acidic variants that are currently not fully understood.

Parental Accompaniment in Operating Rooms Reduces Child Anxiety.

BACKGROUND: We believe that parental presence before the induction of anesthesia for surgery among children with a cleft palate/lip would be effective in mitigating their preoperative anxiety. OBJECTIVE: We assessed the states of patients with a cleft palate/lip when their parents accompanied them into operating rooms and clarified their and their parents' cognition using a questionnaire. METHODS: Data were collected via nursing observation when patients and their parents entered the operating room. Furthermore, an anonymous questionnaire was administered to patients and parents after the operation regarding their feelings about parental presence in the operating room. RESULTS: In total, nine patients cried when they entered the surgical room. Furthermore, six patients and three parents reported preoperative anxiety. In addition, eight patients agreed that they were satisfied with the presence of their parents before induction. CONCLUSION: Approximately half of the patients cried. However, the presence of parents before the induction of anesthesia was effective in reducing anxiety among most patients and their parents.

The infrapatellar plica of the knee: analysis of relationship with femoral trochlear chondrosis using radiographs and 3.0-T MRI.

OBJECTIVE: To assess the relationship of the infrapatellar plica (IPP) with femoral trochlear chondrosis (FTC) using radiographs and 3.0-T MRI. MATERIALS AND METHODS: Four hundred eighty-three knees of 476 patients undergoing radiography and MRI were reviewed, and 280 knees of 276 patients were included. We performed a comparison of the frequency of the IPP between men and women, and that of FTC and chondromalacia patella between knees with and without the IPP. In knees with the IPP, we analyzed the correlation between FTC and sex, age, laterality, Insall-Salvati ratio (ISR), femoral sulcus angle, tilting angle, height of insertion of the IPP to Hoffa's fat pad, and width of the IPP. RESULTS: The IPP was found in 192 of 280 knees (68.6%) overall and was more common in men than in women (100 of 132 [75.8%], 92 of 148 [62.2%], p = 0.01). FTC was observed in 26 of 280 (9.3%) and was only in knees with the IPP (knees with the IPP: 26 of 192 [13.5%], knees without the IPP: 0 of 88 [0%], p < 0.001). In knees with the IPP, ISR was significantly greater in knees with FTC (p = 0.002). ISR was the only significant factor associated with FTC (odds ratio: 2.87, 95% confidence interval: 1.14, 7.22, p = 0.03), and the cutoff value of ISR for FTC was > 1.00 with sensitivity of 69.2% and specificity of 63.9%. CONCLUSION: Presence of the IPP combined with ISR > 1.00 was correlated with FTC.

Viscosity increase/gelation of therapeutic IgG monoclonal antibodies induced by Zn2+: One possible root cause of clogging of staked-in-needle prefilled syringes.

We investigated the elution of zinc ions (Zn2+) from the elastomer of rigid needle shields (RNS) attached to staked-in-needle prefilled syringes (SIN-PFS) and the physicochemical impacts of Zn2+ on therapeutic IgG monoclonal antibody (mAb) solutions. The elution of metal ions from typical RNS elastomer under realistic buffer and storage conditions was investigated by inductively coupled plasma-mass spectrometry. Among the metal ions examined, only Zn2+ was detected. The elution of Zn2+ from RNS elastomer was found to be buffer-dependent. We investigated the influence of Zn2+ on the viscosity of seven mAb solutions at 180 mg/mL. The effect of Zn2+ clearly depended on antibody type. Drastic increases in viscosity or gelation were observed in four out of the seven mAbs. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) showed the effect of Zn2+ on mAb viscosity was explained by the colloidal destabilization of mAb solutions. Thus, Zn2+ leaching from RNS elastomer may possibly increase viscosity or cause gelation, and consequently cause possible needle clogging during long-term storage. DLS and SAXS can predict reactivity of mAbs to Zn2+, and require only small amounts of samples. This makes it possible to predict compatibility with RNS elastomer and evaluate needle clogging risk in SIN-PFSs in the early stages of mAb development.

Stent graft placement guided by intravascular ultrasound for subclavian artery dissecting aneurysm.

Subclavian artery dissecting aneurysm is relatively rare and can be caused by traumatic, nontraumatic, and iatrogenic etiologies. Surgical management of subclavian artery dissecting aneurysm has been sparsely reported. Recently, due advances in endovascular techniques making them less invasive, these approaches have become more standard as treatments. Subclavian artery dissecting aneurysm management usually depends on whether there is ischemia of the tissues supplied by the subclavian artery. Furthermore, treatment strategies depend on which section of the artery is involved. In particular, treatment is difficult if the dissecting aneurysm has branching vessels. In this case report, we show that endovascular repair using a covered stent graft is a promising approach to repair a subclavian artery dissecting aneurysm. In this case, the stent graft was highly effective, and follow-up examinations showed good patency of the subclavian artery. Additional use of IVUS (Volcano Inc.; Rancho Cordova, CA, USA) is helpful to obtain the precise location of the true lumen of a dissecting aneurysm.

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Small-scale screening method for low-viscosity antibody solutions using small-angle X-ray scattering.

In this study, we investigated the concentration range in which self-association starts to form in humanized IgG monoclonal antibody (mAb) solutions. Furthermore, on the basis of the results, we developed a practical method of screening for low-viscosity antibody solutions by using small-angle X-ray scattering (SAXS) measurements utilizing small quantities of samples. With lower-viscosity mAb3, self-association was not detected in the range of 1-80mg/mL. With higher-viscosity mAb1, on the other hand, self-association was detected in the range of 10-20mg/mL and was clearly enhanced by a decrease in temperature. The viscosities of mAb solutions at 160, 180, and 200mg/mL at 25°C quantitatively correlated very well with the particle size parameters obtained by SAXS measurements of mAb solutions at 15mg/mL at 5°C. The quantity of mAb sample required for the SAXS measurements was only 0.15mg, which is about one-hundredth of that required for actual viscosity measurements at a high concentration, and such quantities could be available even at an early stage of development. In conclusion, the SAXS analysis method proposed in this study is a valuable tool for the development of concentrated mAb therapeutics with high manufacturability and high usability for subcutaneous injection.

Quantitative Correlation between Viscosity of Concentrated MAb Solutions and Particle Size Parameters Obtained from Small-Angle X-ray Scattering.

PURPOSE: To investigate the relationship between viscosity of concentrated MAb solutions and particle size parameters obtained from small-angle X-ray scattering (SAXS). METHODS: The viscosity of three MAb solutions (MAb1, MAb2, and MAb3; 40-200 mg/mL) was measured by electromagnetically spinning viscometer. The protein interactions of MAb solutions (at 60 mg/mL) was evaluated by SAXS. The phase behavior of 60 mg/mL MAb solutions in a low-salt buffer was observed after 1 week storage at 25°C. RESULTS: The MAb1 solutions exhibited the highest viscosity among the three MAbs in the buffer containing 50 mM NaCl. Viscosity of MAb1 solutions decreased with increasing temperature, increasing salt concentration, and addition of amino acids. Viscosity of MAb1 solutions was lowest in the buffer containing histidine, arginine, and aspartic acid. Particle size parameters obtained from SAXS measurements correlated very well with the viscosity of MAb solutions at 200 mg/mL. MAb1 exhibited liquid-liquid phase separation at a low salt concentration. CONCLUSIONS: Simultaneous addition of basic and acidic amino acids effectively suppressed intermolecular attractive interactions and decreased viscosity of MAb1 solutions. SAXS can be performed using a small volume of samples; therefore, the particle size parameters obtained from SAXS at intermediate protein concentration could be used to screen for low viscosity antibodies in the early development stage.

Establishment of powder dustiness evaluation method by dustmeter with small amount of pharmaceutical ingredients.

The ratio of high potent materials in the new chemical entities has recently increased in the pharmaceutical industry. Generally, most of them are highly hazardous, but there is little toxicity information about the active pharmaceutical ingredients in the early development period. Even if their handling amount is quite small, the dustiness of high potent powder generated in the manufacturing process has an important impact on worker health; thus, it is important to understand the powder dustiness. The purpose of this study was to establish a method to evaluate the powder dustiness by the consumption of small amount of samples. The optimized measurement conditions for a commercially available dustmeter were confirmed using lactose monohydrate and naproxen sodium. The optimized test conditions were determined: the dustmeter mode, the flow rate, the drum rotation speed, the total measurement time, and sample loaded weight were type I mode, 4 L/min, 10 rpm, 1 min and 1-10 g , respectively. The setup conditions of the dustmeter are considerably valuable to pharmaceutical industries, especially, at the early development stage and especially for expensive materials, because the amount of air-borne dust can be evaluated with accuracy by the consumption of small amount of samples.

Thermodynamic and fluorescence analyses to determine mechanisms of IgG1 stabilization and destabilization by arginine.

PURPOSE: To investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg). METHODS: The effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence. RESULTS: Arg monohydrochloride (Arg-HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg-HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg-HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg-HCl. CONCLUSIONS: Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

O-linked glucosylation of a therapeutic recombinant humanised monoclonal antibody produced in CHO cells.

An unpredictable modification of a therapeutic recombinant humanised monoclonal antibody (rh-mAbX) produced using CHO cells was found. LC/MS analysis of rh-mAbX indicated the presence of heterogeneity in the light chain with a corresponding mass shift of 162 Da compared to the theoretical mass. To characterise the heterogeneity, that is, the attached moiety, several analyses were performed. Peptide mapping of rh-mAbX indicated that the attached moiety was located in the amino acid sequence from Leu20 to Lys45, which is a part of the variable region of the light chain. The peptide was efficiently purified in two-steps by RP-HPLC by utilising two different types of RP columns. N-terminal sequencing and LC/MS/MS analysis of the peptide suggested that Ser29 of the light chain was the modification site, and that the attached moiety was a single O-linked hexose. HPAEC-PAD analysis following ß-elimination indicated the presence of an O-linked glucose in the modified peptide. Monosaccharide composition analysis after acid hydrolysis supported this result. The content of antibodies containing this species was determined to be approximately 10% by Lys-C peptide mapping detected at 280 nm. Thus, this study demonstrated the formation of a unique O-linked glucosylation posttranslational modification in a recombinant humanised monoclonal antibody produced in CHO cells.

C-terminal modification of monoclonal antibody drugs: amidated species as a general product-related substance.

Twelve therapeutic mAbs, comprising 10 IgG1s and 2 IgG4s, were analyzed by a peptide mapping technique to detect and quantify C-terminal modifications. C-terminal amidated structures were found in 8 out of the 12 mAbs. An in vitro study using a commercially available peptidylglycine alpha-amidating monooxygenase (PAM) revealed that both IgG1 and IgG4 can be substrates for PAM. This study showed that C-terminal amidation is a general C-terminal modification on the heavy chains of therapeutic mAbs and that C-terminal amidation of mAbs can be catalyzed by a certain PAM(s) in the Chinese hamster ovary (CHO) cells that are widely used for manufacturing therapeutic mAbs.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

VH/VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody.

Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V(H)/V(L) (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V(H)/V(L) interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V(H)/V(L) association and inhibit the undesired V(H)/V(L) association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V(H)/V(L) interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V(H)/V(L) interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.

Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H(2)(18)O.

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are spontaneous and common alterations occurring in pharmaceutical protein drugs in solution. Because those reactions may cause functional changes, it is important to identify the product-related substances, especially when biopharmaceuticals are under development. In this study, we used H(2)(18)O to identify Asn deamidation and Asp isomerization sites on a recombinant humanized monoclonal antibody (mAb) by using high-performance liquid chromatography-mass spectrometry (HPLC-MS). This strategy takes advantage of reactions whereby (18)O is incorporated into the protein molecule. The mAb was lyophilized and reconstituted in H(2)O or H(2)(18)O, followed by incubation at 50 degrees C for 1 month. Samples were reduced/carboxymethylated and digested by trypsin and then subjected to HPLC-MS and HPLC-tandem mass spectrometry (MS/MS) analysis. Among all of the peptide fragments analyzed, there were two in which deamidation and/or isomerization was observed. In one peptide fragment, an obvious mass shift ( approximately 3Da) at Asn was observed in the newly produced peptide when the mAb was incubated in H(2)(18)O, whereas it was barely feasible to identify this mass shift in H(2)O. In the other peptide fragment, isomerization of Asp was identified after incubation in H(2)(18)O, although it was impossible to distinguish when using H(2)O. By means of this procedure, identification of deamidation and isomerization sites can be accomplished easily even when they are difficult or impossible to detect by the usual peptide mapping.

Tramadol produces outward currents by activating mu-opioid receptors in adult rat substantia gelatinosa neurones.

1. An action of a tramadol metabolite, mono-O-dimethyl-tramadol (M1), on substantia gelatinosa (SG) neurones in adult rat spinal cord slices was examined by using the whole-cell patch-clamp technique. 2. In 41% of the neurones examined, superfusing M1 produced an outward current at -70 mV; this response reversed at a potential close to the equilibrium potential for K(+). M1 current hardly declined and persisted for >30 min after its washout. 3. M1 current correlated in amplitude with current produced by mu-opioid receptor agonist DAMGO in the same neurone, and largely reduced in amplitude in the presence of mu-opioid receptor antagonist CTAP but not alpha2-adrenoceptor antagonist yohimbine. In a neurone where M1 had no effect on holding currents, noradrenaline produced an outward current at -70 mV. 4. The amplitude of the M1 response, relative to that of the DAMGO response, exhibited an EC(50) value of 300 microM. 5. We conclude that M1 produces a persistent hyperpolarization by activating mu-opioid receptors in adult rat SG neurones. This could contribute to at least a part of pain alleviation produced by tramadol.

Microscopic molecular mobility of amorphous AG-041R measured by solid-state 13C NMR.

PURPOSE: AG-041R is characterized to be stable in amorphous state and difficult to crystallize at normal period of time. In order to investigate the molecular mobility in microscopically, the spin-lattice relaxation time (T1) of AG-041R was investigated by solid-state CP/MAS 13C NMR at temperature below and above glass transition temperature (Tg). METHOD: CP/MAS measurement and T1 measurement were performed by means of 13C NMR, where the measurement temperatures were 60, 70, 80, 100, and 110 degrees C. The spin-lattice relaxation time (T1) of AG-041R was calculated from the relaxation curves. RESULTS: From the analysis of T1 of amorphous AG-041R, it was clarified that all of the carbons did not start moving drastically at Tg and there were some groups of carbon in terms of temperature dependency of T1. One is a type, such as the carbons in benzene ring: their T1 was drastically changed at Tg. On the other hand, T1 of carbonyl carbons gradually decreased, and above Tg their T1 was still higher than that of the other carbons. There was no significant change of T1 in the methyl carbons around Tg. From the study of IR and 1H NMR in solution, the inter- and intramolecular hydrogen bondings between NH and C=O were found in AG-041R. Due to hydrogen bonding, the inter- and/or intramolecular interaction is considered to retain even at supercooled liquid state. CONCLUSION: The structure that contributes glass transition is the main skeleton structure, such as benzene ring, while small group, like methyl, start to move at lower temperature than Tg. On the other hand, for the carbons, such as carbonyl, their structure was restricted by inter- and/or intramolecular interaction, therefore, their molecular mobility was significantly low above Tg.