Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody.

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.

Enhanced glyco-profiling by specific glycopeptide enrichment and complementary monolithic nano-LC (ZIC-HILIC/RP18e)/ESI-MS analysis.

Dedicated and specific sample preparation and adequate chromatographic resolution prior to MS are necessary for comprehensive and site-specific glycosylation analysis to compensate for high heterogeneity of protein glycosylation, low-abundance of specific glycoforms and ion-suppression effects caused by coelution of other peptides. This article describes a scheme for glycopeptide profiling, which comprises HILIC batch enrichment followed by complementary HILIC and RP-LC in 1-D and 2-D approaches. For reproducible and sensitive nano-LC/ESI-MS analysis, we used ZIC-HILIC and RP18e monolithic silica capillaries and assessed their retention characteristics and complementarity for glycopeptide separations. The experiments revealed that pre-enrichment of glycopeptides in combination with LC employing both phases considerably improves site-specific elucidation of glycosylation heterogeneity. Zwitterionic hydrophilic interaction liquid chromatography showed high capability to separate glycopeptides by their glycan composition, which coeluted on RP18e. By varying solvent conditions, retention can be well tuned, and efficient separations were achieved even in absence of any additives like salt or formic acid. RP18e facilitated glycopeptide separations with high peak capacity based on peptide sequence and degree of sialylation. Implementing both orthogonal and complementary phases in 1-D and 2-D LC setups was shown to significantly increase the number of different identified glycoforms and possesses great potential for comprehensive glycoproteomics approaches.

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies.

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.

Immunogenicity, efficacy and adverse events of adalimumab in RA patients.

To assess the immunogenicity of adalimumab, a human anti-TNF-alpha mAb, we evaluated the formation of antibodies to adalimumab, efficacy and adverse events among 15 patients with highly active rheumatoid arthritis. Four patients were treated with adalimumab as monotherapy, and 11 patients with concomitant DMARDs. Disease activity was measured by DAS28. The antibodies were detected by ELISA. Thirteen (87%) patients withdrew from therapy within 45 weeks and overall 13 (87%) patients showed antibodies to adalimumab including 11 patients who withdrew from therapy. In four patients without concomitant DMARDs and in nine patients with concomitant DMARDs, we detected anti-adalimumab antibodies. Overall, five of seven patients with adverse drug reactions and all nine patients with lack of efficacy were associated with the formation of antibodies. Two antibody-positive patients developed an exantheme. The results indicate that adalimumab is, in spite of its fully human sequences, immunogenic and induces antibodies in a high rate of adalimumab-treated patients.

Peripheral INSL3 concentrations decline with age in a large population of Australian men.

The novel peptide hormone insulin-like peptide 3 (INSL3) is a major secretory product of the Leydig cells of the testis, and in adult men is secreted into the blood, giving rise to circulating concentrations ranging from 0.5 to 2.5 ng/mL. We studied a large randomly recruited cohort of 1183 men from South Australia, comparing serum INSL3 concentrations with age, and a variety of endocrine, cognitive and morphological parameters. While INSL3 concentration declines significantly (p < 0.001) and continuously with age from 1.29 +/- 0.47 ng/mL in young men (age 35-44 years) to 0.79 +/- 0.39 ng/mL in the age group 75-80 years, there is no correlation with testosterone or components of the hypothalamo-pituitary-gonadal (HPG) axis, independent of age, nor with any other parameter measured, including thyroid or prostate status and obesity. For men exhibiting normal follicle stimulating hormone (FSH) and high luteinizing hormone (LH) levels, there was a significant inverse correlation with plasma oestradiol. Unilaterally orchidectomized men had INSL3 values intermediate between intact men and anorchid subjects, and showed inverse correlations (p < 0.001) between INSL3 and FSH or LH concentrations, which were independent of age. Taken together, the data show that INSL3 is an independent measure of Leydig cell function (quality and number), which appears to be independent of acute control via the HPG axis. Its decline with age reflects a decline in the properties of the Leydig cell population only, and emphasizes a gonadal component in the age-related decrease in androgen production.