Recombinant Human G-CSF Characterization

Introduction to Recombinant Human G-CSF

Neutropenia, a medical condition distinguished by a decline in neutrophil concentrations under the standard range, approximated at 1.5 ×109/L, manifests itself across a multitude of disease circumstances, incorporating both hereditary disorders and infection. The condition is particularly prevalent in cancer patients subjected to cytotoxic chemotherapy. A notable consequence of neutropenia is a heightened vulnerability to bacterial and subsequent fungal infections, frequently necessitating hospital admission.

One primary therapeutic application of granulocyte-colony stimulating factor (G-CSF) centers on counteracting neutropenia. The administration of G-CSF possesses the capacity to either curtail the duration of neutropenia or preclude it completely. Moreover, treatment by means of G-CSF can facilitate the execution of higher-intensity dosing schedules, possibly enhancing the efficacy of antitumor undertakings.

Fig 1. G-CSF administration results in thrombocytopenia by inhibiting the differentiation of hematopoietic progenitors into megakaryocytes. (Li, Yunqian, et al. 2019)

In recognition of its potential to alleviate neutropenia, especially during or subsequent to chemotherapy, the pharmaceutical agent Neupogen® was authorized for use by the United States Food and Drug Administration in 1991. The medication has, in the intervening years, achieved approval from a multitude of global regulatory bodies.

Primary Structure of G-CSF

Neupogen® is comprised of a 175-amino acid polypeptide chain. When in solution, this FDA-approved drug presents as a monomeric molecule and has a molecular weight of 18,799.94, as deduced from its genomic sequence. Employing N-terminal sequence analysis on purified G-CSF, methionine functions as the singular amino terminus. This insinuates that unlike other proteins, the initiator of protein synthesis in this context does not necessarily experience post-translational processing.

Fig 2. Overlay of wild type G-CSF (PDB Code: 1CD9) with the representative structure of most populated cluster resulted from 100 ns simulation of M1 and M2 mutant. (Mishra, Avinash, et al. 2020)

The bioactive conformation of G-CSF is preserved by the formation of two compact loop structures resultant of two intramolecular disulfide bonds, specifically, Cys37-Cys43 and Cys65-Cys75. A reductive alteration in any of these disulfide bonds can significantly hinder the protein's overall activity. Moreover, Neupogen® incorporates a solo cysteine at the 18th position which, interestingly, is not a prerequisite for its bioactivity.

Complete Amino Acid Sequence

Neupogen® was subjected to in-depth structural characterization to accurately determine its chemical composition. Amino acid analysis and peptide mapping were performed using a variety of analytical techniques and the amino acid sequence was determined. S-carboxymethylated Neupogen® was digested with the endoprotease Glu-C to generate peptide fragments. In addition, the complete protein was digested using chymotrypsin. After digestion, these fragments were separated by reversed-phase high-performance liquid chromatography (HPLC). After cleavage with the endoprotease Glu-C, a typical peptide map of alkylated Neupogen® was drawn, and this method has been the reference for our product characterization tests.

Fig 3. HPLC peptide map of reduced and carboxymethylated Neupogen® after endoproteinase Glu-C digestion. (Pearlman, Rodney, and Y. John Wang, eds. 1996)

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Through the adept utilization of advanced methodologies and revolutionary technologies, Creative Proteomics proffers an extensive repertoire of all-encompassing protein drug characterization services. Our accomplished team of scientists with profound expertise ensures the provision of precise and dependable analyses, facilitating an intricate understanding of the attributes, qualities, and mechanisms of protein drugs. Please do not hesitate to get in touch with us to explore our services further.

References

1. Li, Yunqian, et al. G-CSF administration results in thrombocytopenia by inhibiting the differentiation of hematopoietic progenitors into megakaryocytes. Biochemical Pharmacology. 2019, 169: 113624.
2. Mishra, Avinash, et al. Mechanistic explanation of structural and functional changes induced by methionine mutation in G-CSF protein. Current Research in Biotechnology. 2020, 2: 37-44.
3. Pearlman, Rodney, and Y. John Wang, eds. Formulation, characterization, and stability of protein drugs. Vol. 9. Springer Science & Business Media. 1996.

For research use only, not intended for any clinical use.