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1.
Anal Chem ; 89(13): 7225-7231, 2017 07 05.
Article in English | MEDLINE | ID: mdl-28585810

ABSTRACT

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.


Subject(s)
Deoxyribonuclease I/radiation effects , Histidine/radiation effects , Protein Multimerization/radiation effects , Deoxyribonuclease I/chemistry , Histidine/chemistry , Hydrophobic and Hydrophilic Interactions , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/radiation effects , Ultraviolet Rays , Water/chemistry
2.
Biologicals ; 44(5): 291-305, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27461239

ABSTRACT

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Quality Control , Animals , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology
3.
J Pharm Sci ; 104(2): 368-77, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25175600

ABSTRACT

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/metabolism , Methionine/chemistry , Methionine/metabolism , Receptors, Fc/chemistry , Receptors, Fc/metabolism , Binding Sites , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Oxidation-Reduction , Surface Plasmon Resonance
4.
Anal Chem ; 86(10): 5150-7, 2014 May 20.
Article in English | MEDLINE | ID: mdl-24749737

ABSTRACT

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to a placebo that has no protein. The multidimensional UHPLC-CAD-MS approach provides insights into the heterogeneous stability behaviors of polysorbate 20 subspecies in real-time stability samples of a mAb formulation.


Subject(s)
Antibodies, Monoclonal/chemistry , Excipients/chemistry , Polysorbates/chemistry , Aerosols , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Mass Spectrometry
5.
J Pharm Biomed Anal ; 83: 273-8, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23770782

ABSTRACT

Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ~150 kDa, reduced mAb fragments of ~25-50 kDa and also digested mAb generating numerous peptides of ~0.5-3 kDa. Several long columns packed with fully porous wide-pore sub-2 µm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150 mm length, a mobile phase temperature of 80 °C and a gradient time of around 20 min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50 °C, with a gradient time of 270 min and a column length of 450 mm, a peak capacity of more than 700 was achieved.


Subject(s)
Antibodies, Monoclonal/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Recombinant Proteins/chemistry , Porosity
6.
MAbs ; 4(6): 694-700, 2012.
Article in English | MEDLINE | ID: mdl-23018810

ABSTRACT

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC-MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.


Subject(s)
Immunoglobulin G/metabolism , Immunoglobulin Light Chains/metabolism , Animals , Base Pair Mismatch/genetics , CHO Cells , Codon, Terminator/genetics , Cricetinae , Cricetulus , DNA Mutational Analysis , Glutamine/genetics , Immunoglobulin G/genetics , Immunoglobulin Light Chains/genetics , Mass Spectrometry , Mutation/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Processing, Post-Transcriptional/genetics
7.
Anal Chem ; 84(16): 7112-23, 2012 Aug 21.
Article in English | MEDLINE | ID: mdl-22794164

ABSTRACT

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.


Subject(s)
Antibodies, Monoclonal/chemistry , Cysteine , Immunoglobulin G/chemistry , Recombinant Proteins/chemistry , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/toxicity , Antigens, CD20/immunology , CHO Cells , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Chromatography, Reverse-Phase , Cricetinae , Cricetulus , Humans , Immunoglobulin G/immunology , Immunoglobulin G/toxicity , Mass Spectrometry , Protein Denaturation , Recombinant Proteins/immunology , Recombinant Proteins/toxicity , Sulfhydryl Compounds/chemistry
8.
Anal Chem ; 84(5): 2313-20, 2012 Mar 06.
Article in English | MEDLINE | ID: mdl-22324758

ABSTRACT

Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.


Subject(s)
Antibodies/chemistry , Carbohydrates/chemistry , Glycopeptides/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Amino Acid Sequence , Antibodies/genetics , Antibodies/metabolism , Carbon Isotopes/chemistry , Chromatography, High Pressure Liquid , Glycosylation , Isotope Labeling , Oxidation-Reduction , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
9.
J Chromatogr A ; 1218(31): 5079-86, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21700290

ABSTRACT

In this report, we have demonstrated the isolation and enrichment of charge variants of a monoclonal antibody IgG1 using cation exchange displacement chromatography. We successfully achieved the separation of acidic, main and basic charge variants with high recovery (>70%) and purity (>90%) by using a commercially available stationary phase in conjunction with a commercially available displacer. In addition, we have isolated and enriched a trace methionine-oxidized variant of the monoclonal antibody allowing a secondary means of identification of this variant while providing sufficient enrichment for further analysis, stability tests and potency determination. Further characterization of the displacement trains by SEC indicate the possibility of enrichment of high and low molecular weight species. Glycan analysis of the displacement fractions indicates minimal variation in glycan distribution patterns among a wide spectrum of charge variants. These results provide a case study demonstrating the utility of cation exchange displacement chromatography as a viable approach to isolate and enrich antibody charge variants for enhanced molecular characterization.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Chromatography, Ion Exchange/methods , Immunoglobulin G/isolation & purification , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , CHO Cells , Cricetinae , Cricetulus , Drug Therapy , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/therapeutic use
10.
J Chromatogr A ; 1218(15): 2138-45, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-20950816

ABSTRACT

Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate. The results indicated POE sorbitan was present from 16.0 to 27.6 and 11.1 to 14.5% (w/w) in polysorbate 20 and 80, respectively. The second HPLC method used a reversed-phase stationary phase (Zorbax SB-300 C(8)) with a shallow gradient to separate, identify, and quantify the multiple ester species present in polysorbate. For all lots of polysorbate 20 analyzed, only 18-23% of the material was the expected structure, polyoxyethylenesorbitan monolaurate. Up to 40% and 70% (w/w) di- and triesters were found in polysorbate 20 and polysorbate 80 respectively. Likewise, polyoxyethylenesorbitan monooleate accounted for only 20% of polysorbate 80. A variability of 3-5% was observed for each ester species between multiple lots of polysorbate 20. The reversed-phase method was then used to determine the rate of hydrolysis for each polyoxyethylene sorbitan ester of polysorbate 20 in basic solution at room temperature. Increasing rates of hydrolysis were observed with decreasing aliphatic chain lengths in polysorbate 20.


Subject(s)
Chromatography, Reverse-Phase/methods , Polysorbates/chemistry , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid , Hydrolysis , Light , Scattering, Radiation
11.
J Chromatogr A ; 1215(1-2): 156-60, 2008 Dec 26.
Article in English | MEDLINE | ID: mdl-19036378

ABSTRACT

An HPLC assay requiring no complex sample preparation for the measurement of polysorbate 20 in protein solutions was developed. An on-off chromatography technique was employed involving a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion-exchange and reversed-phase sorbent) to quantify polysorbate 20 in solutions containing >100mg/mL of protein. With 2% formic acid mobile phase, proteins are typically positive charged and are not retained because of electrostatic repulsions from the quaternary amine in the mixed-mode resin. Other formulation components elute in void volume because of their hydrophilicity. Hydrophobic polysorbate 20 is retained, eluted with a step gradient and quantified as a single peak using an evaporative light scattering detector. The performance of the assay is evaluated according to International Conference on Harmonisation (ICH) guidelines and shown to be suitable for polysorbate quantitation. Accuracy (96-108%) and repeatability (2.3% RSD) were demonstrated using protein samples spiked with polysorbate 20. This method was used to accurately measure polysorbate 20 in at least 25 different protein solutions spanning a wide range of formulations. Although the majority of the data reported here target polysorbate 20, this methodology can also be used to assay other common non-ionic surfactants such as polysorbate 80, Brij, Igepal, and Triton X-100.


Subject(s)
Chromatography, High Pressure Liquid/methods , Polysorbates/analysis , Proteins/chemistry , Light , Reproducibility of Results , Scattering, Radiation , Sensitivity and Specificity , Solutions
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