Chemical modifications of therapeutic proteins induced by residual ethylene oxide.

Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics.

LC-MS/MS peptide mapping with automated data processing for routine profiling of N-glycans in immunoglobulins.

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

IgG2 disulfide isoform conversion kinetics.

Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.

Nutrition challenges in a patient with sinusoidal obstructive syndrome following an allogeneic stem cell transplant: a case study.

Nutrition management of patients undergoing a hematopoietic stem cell transplant (HSCT) can be challenging. Fluid, macronutrient, and micronutrient needs are often altered due to a variety of therapy-associated complications and changes in metabolism. Sinusoidal obstructive syndrome (SOS) is a complication characterized by fluid retention, ascites, and painful hepatomegaly that may complicate provision of adequate nutrition to HSCT patients. The nutrition implications and interventions in a patient who developed SOS following an allogeneic matched unrelated donor (Allo/MUD) HSCT are reviewed in the case report.

Biochemical assessment of erythropoietin products from Asia versus US Epoetin alfa manufactured by Amgen.

We compared the physical and chemical properties of purported copies of recombinant human erythropoietin (rHuEPO) purchased from Korea, China, and India with the innovator product, Epoetin alfa, manufactured by Amgen Inc. The products were characterized for similarity in the types of glycoforms present, the relative degree of unfolding, in vitro potency, presence of covalent aggregates, and presence of cleavage products using established analytical methods. All products were different from Epoetin alfa (Epogen). The purported copies of rHuEPO from Korea, India, and China contained more glycoforms and other impurities. The in vitro relative potency varied for each product when based on the labeled concentration, while the concentration based on ELISA analysis brought the relative potency, for most products closer to 100%. These data emphasize potential biochemical discrepancies resulting from different cell lines and manufacturing processes. Concentrations varied within products and did not always match the information provided on the product label. As it is not possible to reliably correlate such biochemical discrepancies to clinical consequences, or the lack thereof, these data support the need for extensive preclinical testing and clinical testing of all investigational products as not all safety and efficacy aspects can be assessed during preclinical evaluation.