Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines.

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a ß1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLe(x) epitope was only detected in Dynepo.

Peptide mapping with liquid chromatography using a basic mobile phase.

A new peptide mapping with liquid chromatography (LC) using an ammonia-containing basic mobile phase was reported. As compared with a method under a traditional acidic condition with a mobile phase containing trifluoroacetic acid (TFA) or formic acid (FA), the new method exhibited excellent overall performance: it was advantageous over the TFA method in terms of the ultraviolet (UV) and mass spectrometry (MS) sensitivities and the sequence coverage for a tryptic map; it was superior to the FA method in terms of the UV sensitivity, the sequence coverage and the separation capacity. Due to a significant difference in the chromatographic selectivity, several important peptide mapping applications that were sometimes difficult to be conducted previously could now be carried out using the new method. For example, the baseline separation of peptides from the corresponding deamidated products could be achieved with confidence using the new method, a critical pre-requisite for definitive identification and quantification of the deamidation products with LC/MS. No on-column deamidation was observed with the conditions used for the separation. Complementary and confirmative information about a protein could be obtained by running its proteolytic digest under both the basic and acidic conditions.

Challenges in the development of high protein concentration formulations.

Development of formulations for protein drugs requiring high dosing (in the order of mg/kg) may become challenging for solubility limited proteins and for the subcutaneous (SC) route with <1.5 mL allowable administration volume that requires >100 mg/mL protein concentrations. Development of high protein concentration formulations also results in several manufacturing, stability, analytical, and delivery challenges. The high concentrations achieved by small scale approaches used in preformulation studies would have to be confirmed with manufacturing scale processes and with representative materials because of the lability of protein conformation and the propensity to interact with surfaces and solutes which render protein solubilities that are dependent on the process of concentrating. The concentration dependent degradation route of aggregation is the greatest challenge to developing protein formulations at these higher concentrations. In addition to the potential for nonnative protein aggregation and particulate formation, reversible self-association may occur, which contributes to properties such as viscosity that complicates delivery by injection. Higher viscosity also complicates manufacturing of high protein concentrations by filtration approaches. Chromatographic and electrophoretic assays may not accurately determine the non-covalent higher molecular weight forms because of the dilutions that are usually encountered with these techniques. Hence, techniques must be used that allow for direct measurement in the formulation without substantial dilution of the protein. These challenges are summarized in this review.

Asparagine deamidation in recombinant human lymphotoxin: hindrance by three-dimensional structures.

The chemical stability of recombinant human lymphotoxin (rhLT) was evaluated at pH 7, 9, and 11 and 40 degrees C using quantitative tryptic map and urea-IEF methods. Degradation products were characterized by mass spectrometry. The stability of denatured rhLT protein was also evaluated to elucidate the effects of three-dimensional structures on Asn deamidation in rhLT. Two sites that underwent Asn deamidation were identified in rhLT, Asn(19) and Asn(40)-Asn(41). At pH 11 and 40 degrees C, deamidation at Asn(19) and Asn(40)-Asn(41) had half-lives of 14 +/- 4 and 80 +/- 24 days, respectively. Upon denaturation, 31- and ninefold acceleration in the degradation rates was observed at the Asn(19) and Asn(40)-Asn(41) sites, respectively. The rate of Asn(19) degradation in denatured rhLT was comparable to that of the model peptide possessing the same primary sequence as the Asn(19)-containing region in rhLT. Analysis of the rhLT crystal structure revealed that both Asn deamidation sites were located in beta-turn structures with extensive hydrogen-bonding networks created with nearby residues in the tertiary structures. The results suggested that these tertiary and secondary structures, if held true in solution, were probably responsible for the stabilization of Asn in the native rhLT protein by reducing flexibility, thus preventing adoption of the favorable conformation required for cyclic-imide formation.