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1.
Anal Sci ; 38(5): 739-747, 2022 May.
Article in English | MEDLINE | ID: mdl-35297021

ABSTRACT

The multi-attribute method has been recognized as an elegant quantification tool for post-translational modifications (PTMs) of therapeutic proteins, since it can evaluate several attributes spontaneously and site-specifically. Here, the abundance of PTMs calculated by three different types of formula were compared and there was little difference among the results. For the method evaluation, two different kinds of peptides were used as internal standards (ISs) and one of the IS was used as the "standard peak" to define the signal strength of MS. They are also used for system suitability testing to verify whether the condition or sensitivity of mass spectrometry are high enough to evaluate the minor components by confirming the recovery rate of one IS to the another. This system is beneficial that since we have defined the limit of quantification as a certain ratio to IS, consistent MS intensity is applied as the threshold across all detected peaks.


Subject(s)
Antibodies, Monoclonal , Protein Processing, Post-Translational , Mass Spectrometry , Peptides , Quality Control
2.
J Pharm Sci ; 110(10): 3367-3374, 2021 10.
Article in English | MEDLINE | ID: mdl-34089708

ABSTRACT

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.


Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents, Immunological , Chromatography, Gel , Complementarity Determining Regions , Immunoglobulin Fc Fragments
3.
Anal Sci ; 35(12): 1333-1340, 2019 Dec 10.
Article in English | MEDLINE | ID: mdl-31423004

ABSTRACT

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant ß-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and ß-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.


Subject(s)
Antithrombins/chemistry , Antithrombins/metabolism , Genetic Engineering/methods , Oligosaccharides/chemistry , Plasma/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Fucosyltransferases/deficiency , Fucosyltransferases/genetics , Gene Knockout Techniques , Glycosylation , Humans , Recombinant Proteins/genetics
4.
Pharm Res ; 33(4): 994-1002, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26694753

ABSTRACT

PURPOSE: In biopharmaceutical development, information regarding higher-order structure (HOS) is important to verify quality and characterize protein derivatives. In this study, we aimed to characterize the association between HOS and pharmacokinetic property of a stress-exposed monoclonal antibody (mAb). METHODS: Purity, primary structure, thermal stability, and HOS were evaluated for mAbs exposed to heat, photo-irradiation, and chemical oxidation. To investigate conformation of stress-exposed mAbs, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was utilized. RESULTS: No distinct difference in secondary or tertiary structure between stress-exposed and non-stressed samples was found by conventional spectroscopic techniques. In binding activity with the neonatal Fc receptor (FcRn), however, a marked decline was observed for force-oxidized mAb and a slight decline was observed for heat- and photodegraded mAbs. Using differential scanning calorimetry, a change in thermal stability was observed in the CH2 domain for all the stress-exposed samples. Using HDX-MS analyses, individual regions with altered conformation could be identified for heat-degraded and force-oxidized samples. CONCLUSIONS: These findings indicate that comprehensive study is important for detecting conformational changes and helpful for predicting biophysical property, and that the evaluation of HOS using several analytical techniques is indispensable for confirming biopharmaceutical quality.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Histocompatibility Antigens Class I/immunology , Receptors, Fc/immunology , Hot Temperature , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Oxidation-Reduction , Photolysis , Protein Conformation , Protein Stability
5.
J Biochem ; 156(6): 345-52, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25147193

ABSTRACT

To investigate the relationship between conformational stability, reversibility of denaturation and aggregation of protein, we determined the conformation, melting temperature (Tm), and reversibility of heat-induced denaturation of α-1-acid glycoprotein (AGP) in aqueous solutions at various pH values using circular dichroism (CD) and differential scanning microcalorimetry. To quantitate and characterize heat-induced AGP aggregation under the same pH conditions, solutions of AGP were incubated at 50°C and then analysed by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CD and SEC in the presence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The conformational stability of AGP was reduced at lower pH, whereas the reversibility of protein denaturation was reduced at higher pH. AGP formed some large non-covalent aggregates during incubation at lower pH, whereas incubation at higher pH tended to cause the formation of dimer species without the formation of large aggregates. These results indicated that lower conformational stability was related to the formation of non-covalent large aggregates, whereas reduced reversibility was related to dimer formation. Thus, evaluating both conformational stability and reversibility is necessary for developing optimal formulations and to predict the kinds of aggregates that will be induced during protein storage.


Subject(s)
Orosomucoid/chemistry , Protein Conformation , Protein Stability , Calorimetry, Differential Scanning , Chromatography, Gel , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Protein Aggregates , Protein Denaturation , Transition Temperature
6.
J Am Soc Mass Spectrom ; 25(10): 1747-54, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25070584

ABSTRACT

Information on the higher-order structure is important in the development of biopharmaceutical drugs. Recently, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has been widely used as a tool to evaluate protein conformation, and unique automated systems for HDX-MS are now commercially available. To investigate the potential of this technique for the prediction of the activity of biopharmaceuticals, granulocyte colony stimulating factor (G-CSF), which had been subjected to three different stress types, was analyzed using HDX-MS and through comparison with receptor-binding activity. It was found that HDX-MS, in combination with ion mobility separation, was able to identify conformational changes in G-CSF induced by stress, and a good correlation with the receptor-binding activity was demonstrated, which cannot be completely determined by conventional peptide mapping alone. The direct evaluation of biological activity using bioassay is absolutely imperative in biopharmaceutical development, but HDX-MS can provide the alternative information in a short time on the extent and location of the structural damage caused by stresses. Furthermore, the present study suggests the possibility of this system being a versatile evaluation method for the preservation stability of biopharmaceuticals.


Subject(s)
Deuterium Exchange Measurement/methods , Enzyme-Linked Immunosorbent Assay/methods , Granulocyte Colony-Stimulating Factor/chemistry , Mass Spectrometry/methods , Recombinant Proteins/chemistry , Amino Acid Sequence , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Molecular Sequence Data , Protein Binding , Protein Conformation , Recombinant Proteins/metabolism
7.
J Biochem ; 155(1): 63-71, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24155259

ABSTRACT

Intermolecular interactions and conformation in dimer species of Palivizumab, a monoclonal antibody (IgG1), were investigated to elucidate the physical and chemical properties of the dimerized antibody. Palivizumab solution contains ∼1% dimer and 99% monomer. The dimer species was isolated by size-exclusion chromatography and analysed by a number of methods including analytical ultracentrifugation-sedimantetion velocity (AUC-SV). AUC-SV in the presence of sodium dodecyl sulphate indicated that approximately half of the dimer fraction was non-covalently associated, whereas the other half was dimerized by covalent bond. Disulphide bond and dityrosine formation were likely to be involved in the covalent dimerization. Limited proteolysis of the isolated dimer by Lys-C and mass spectrometry for the resultant products indicated that the dimer species were formed by Fab-Fc or Fab-Fab interactions, whereas Fc-Fc interactions were not found. It is thus likely that the dimerization occurs mainly via the Fab region. With regard to the conformation of the dimer species, the secondary and tertiary structures were shown to be almost identical to those of the monomer. Furthermore, the thermal stability turned out also to be very similar between the dimer and monomer.


Subject(s)
Antibodies/metabolism , Immunoglobulin G/metabolism , Antibodies/chemistry , Chromatography, Gel , Circular Dichroism , Dimerization , Electrophoresis, Polyacrylamide Gel , Fluorescence , Mass Spectrometry , Protein Conformation , Ultracentrifugation
8.
J Biochem ; 140(1): 49-56, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16877768

ABSTRACT

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.


Subject(s)
Granulocyte Colony-Stimulating Factor/chemistry , Methanol/pharmacology , Anilino Naphthalenesulfonates/chemistry , Circular Dichroism , Dose-Response Relationship, Drug , Filgrastim , Hot Temperature , Methanol/administration & dosage , Protein Denaturation , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , Recombinant Proteins , Spectrometry, Fluorescence , Spectrophotometry, Infrared
9.
J Biochem ; 139(1): 41-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16428318

ABSTRACT

This paper reports the effect of ionic strength on the process of thermal unfolding of recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) at acid pH. We previously reported that the protein aggregates were formed at the highest temperature at pD 2.1 in the pD range of 5.5-2.1 and that the aggregation proceeded a little at pD 2.1 because of the strong repulsive interaction between the unordered structures that play the role of a precursor for the aggregation. In the present study temperature-dependent IR spectra and far-UV CD spectra were measured for rmethuG-CSF in aqueous solutions containing various concentrations of NaCl at acid pH. Second derivative and curve-fitting analysis were performed to examine the obtained IR spectra. The results revealed that the structure of rmethuG-CSF becomes less stable with increasing ionic strength at all pDs investigated (pD 2.1, 2.5, and 4.0). We have also demonstrated that, at pD 2.1, the temperature at which the protein aggregation starts becomes lower and that the amount of the aggregates becomes larger with the addition of NaCl. This is probably because the addition of NaCl masks the repulsive electrostatic interaction between the unordered structures.


Subject(s)
Granulocyte Colony-Stimulating Factor/metabolism , Osmolar Concentration , Protein Denaturation , Circular Dichroism , Filgrastim , Granulocyte Colony-Stimulating Factor/chemistry , Humans , Recombinant Proteins , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Static Electricity
10.
J Biochem ; 137(3): 265-71, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15809327

ABSTRACT

Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.


Subject(s)
Granulocyte Colony-Stimulating Factor/chemistry , Protein Denaturation/physiology , Protein Folding , Drug Stability , Fourier Analysis , Hot Temperature , Humans , Hydrogen-Ion Concentration , Recombinant Proteins , Spectrophotometry, Infrared
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