NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin-like growth factor-1 (mecasermin).

RATIONALE: Mecasermin is used to treat elevated blood sugar as well as growth-hormone-resistant Laron-type dwarfism. Mecasermin isolated from inclusion bodies in extracts of E. coli must be refolded to acquire sufficient activity. However, there is no rapid analytical method for monitoring refolding during the purification process. METHODS: We prepared mecasermin drug product, in-process samples during the oxidation of mecasermin, forced-reduced mecasermin, and aerially oxidized mecasermin after forced reduction. Desalted mecasermin samples were analyzed using MALDI-ISD. The peak intensity ratio of product to precursor ion was determined. The charge-state distribution (CSD) of mecasermin ions was evaluated using ESI-MS coupled with SEC-mode HPLC. The drift time and collision cross-sectional area (CCS) of mecasermin ions were evaluated using ESI-IMS-MS coupled with SEC-mode HPLC. RESULTS: MALDI-ISD data, CSD values determined using ESI-MS, and the CCS acquired using ESI-IMS-MS revealed the relationship between the folded and unfolded proteoforms of forced-reduced mecasermin and aerially oxidized mecasermin with the free-SH:protein ratio of mecasermin drug product. The CCS area, which is determined using ESI-IMS-MS, provided proteoform information through rapid monitoring (<2 min) of in-process samples during the manufacture of mecasermin. CONCLUSIONS: ESI-IMS-MS coupled with SEC-mode HPLC is a rapid and robust method for analyzing the free-SH:protein ratio of mecasermin that allows proteoform changes to be evaluated and monitored during the oxidation of mecasermin. ESI-IMS-MS is applicable as a process analytical technology tool for identifying the "critical quality attributes" and implementing "quality by design" for manufacturing mecasermin.

Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction.

An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 µM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.

Retention of glycopeptides analyzed using hydrophilic interaction chromatography is influenced by charge and carbon chain length of ion-pairing reagent for mobile phase.

Characterization of the glycans of glycoproteins is essential for the development and production of biologics. Numerous methods are available for analyzing the glycans of glycoproteins directly and labeled glycans. Nevertheless, glycopeptides are difficult to resolve because of their exceptional complexity and the microheterogeneity of glycans. These properties represent technical challenges to efforts to insure the accurate characterization of biopharmaceuticals to comply with regulatory requirements. Therefore, we investigated the retention behavior of peptides and glycopeptides in hydrophilic interaction chromatography-mode HPLC in the presence of ion-pairing reagents. Anionic ion-pairing reagents decreased the retention times of glycopeptides and improved resolution in the presence of higher concentrations or hydrophobicities of ion-pairing reagent. Anionic ion-pairing reagents increased retention times of larger glycans because of their increased hydrophilicity. In contrast, in the presence of cationic ion-pairing reagents, the retention times of glycopeptides with greater numbers of sialic acid residues decreased. It is appropriate to add an anionic ion-pairing reagent to the mobile phase for good separation of glycopeptides. The collision cross-sectional area values of glycopeptides determined using electrospray ionization-ion mobility spectrometry-mass spectrometry correlated with retention times. These findings support the implementation of hydrophilic interaction chromatography-mode HPLC to improve the characterization of glycosylated biopharmaceuticals.

Highly sensitive glycosylamine labelling of O-glycans using non-reductive ß-elimination.

When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.

Active species for Ce(IV)-induced hydrolysis of phosphodiester linkage in cAMP and DNA.

The hydrolysis of cyclic adenosine 3',5'-monophosphate and 2'-deoxythymidylyl(3'-5')2'-deoxythymidine by Ce(NH4)2(NO3)6 was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce2(IV) (OH)4]4+ , showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce2(IV) (OH)4]4+ cluster are bridged by two OH residues. Upon the complex formation with H2 PO4- (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results.

Complete nucleotide sequence of the S10-spc operon of phytoplasma: gene organization and genetic code resemble those of Bacillus subtilis.

An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma. Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon. This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously. The operon has a context of 5'-rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, rps17, rpl14, rpl24, rpl5, rps14, rps8, rpl6, rpl18, rps5, rpl30, rpl15, SecY-3', and is composed of 21 ribosomal protein subunit genes and a SecY protein translocase subunit gene. Resembling Bacillus, this operon contains an rpl30 gene that other mollicutes (Mycoplasma genitalium, M. pneumoniae, and M. pulmonis) lack. A phylogenetic tree based on the rps3 sequence showed that phytoplasmas are phylogenetically closer to acholeplasmas and bacillus than to mycoplasmas. In the S10-spc operon, translation may start from either a GTG codon or an ATG codon, and stop at a TGA codon, as has been reported for acholeplasmas and bacillus. However, in mycoplasmas, GTG was found as a start codon, and TGA was found not as a stop codon, but instead as a tryptophan codon. These data derived from the gene organization, and the genetic code deviation support the hypothesis that phytoplasmal genes resemble those of acholeplasmas and Bacillus more than those of other mollicutes.