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Published on: January 7, 2016
Development of Protein-Specific Analytical Methodologies to Evaluate Compatibility of Recombinant Human
Nazila Salamat-Miller1, Wanlu Qu2, Jennifer S Chadwick2
1Process Development, Formulation Development, Takeda, Lexington, MA, USA.
Insights
Recombinant human insulin-like growth factor-1/binding protein-3 (rhIGF-1/rhIGFBP-3) compatibility was assessed with neonatal medications. Furosemide was incompatible, while ampicillin formed adducts, and caffeine citrate showed no degradation.
Area of Science:
- Biochemistry and Pharmacology
- Neonatal Medicine
- Analytical Chemistry
Background:
- Recombinant human insulin-like growth factor-1 and insulin-like growth factor binding protein-3 (rhIGF-1/rhIGFBP-3) is an investigational therapy for preventing complications in preterm infants.
- Administration involves continuous intravenous infusion, necessitating co-administration with other neonatal medications.
- Evaluating the stability and compatibility of rhIGF-1/rhIGFBP-3 under these conditions is crucial for safe and effective use.
Purpose of the Study:
- To develop and validate sensitive analytical methods for quantifying rhIGF-1/rhIGFBP-3 and its potential degradants.
- To assess the compatibility of rhIGF-1/rhIGFBP-3 with 24 commonly used neonatal medications at low concentrations.
- To identify any degradation, oxidation, aggregation, or adduct formation upon co-administration.
Main Methods:
- Development of protein-specific analytical methodologies including reversed-phase high-performance liquid chromatography (RP-HPLC) and size-exclusion ultra-performance liquid chromatography (SE-UPLC).
- Coupling of chromatography with mass spectrometric detection for sensitive analysis of rhIGF-1/rhIGFBP-3, its components, and degradants.
- Quantification of rhIGF-1/rhIGFBP-3 in admixtures with 24 neonatal medications, with optimization using furosemide, caffeine citrate, and ampicillin.
Main Results:
- Analytical methods demonstrated good reproducibility, linearity, and low limits of detection/quantitation (3.1 μg/mL).
- No significant increase in degradation, oxidation, or aggregation of rhIGF-1/rhIGFBP-3 was observed with caffeine citrate.
- Ampicillin admixtures showed lower mass recovery, likely due to adduct formation, while furosemide was found to be physically incompatible.
Conclusions:
- The developed analytical methodologies are suitable for assessing protein modifications of rhIGF-1/rhIGFBP-3 under clinical co-administration conditions.
- Compatibility findings indicate that caffeine citrate is suitable for co-administration, while ampicillin requires further investigation due to adduct formation.
- Furosemide is not compatible with rhIGF-1/rhIGFBP-3, necessitating separate administration or alternative therapies.
Abstract:
The protein complex of recombinant human insulin-like growth factor-1 and insulin‑like growth factor binding protein‑3 (rhIGF-1/rhIGFBP-3; mecasermin rinfabate), is an investigational product for the prevention of complications of prematurity. Delivery of rhIGF-1/rhIGFBP-3 is by continuous central line intravenous infusion in preterm infants until endogenous IGF-1 production begins. Protein-specific analytical methodologies were developed to evaluate the compatibility of rhIGF-1/rhIGFBP-3 at low protein concentrations (∼2.5-10 μg/mL) expected when co-administered with other required medications in the NICU. Highly sensitive detection of the biologic potential degradants (fragments) and/or molecular modifications (oxidized species, aggregates) required the use of reversed-phase high-performance liquid chromatography and size-exclusion ultra-performance liquid chromatography coupled with mass spectrometric detection. We report on the quantification of rhIGF-1/rhIGFBP-3, its components and degradants, to a limit of quantitation of 3.1 μg/mL upon mixing with 24 commonly administered neonatal medications. Methods developed for the rhIGF-1/rhIGFBP-3 admixtures, optimized in studies with furosemide, caffeine citrate and ampicillin, demonstrated good reproducibility, linearity, and limit of detection/quantitation. Using these methods, no increase in degradation of rhIGF-1/rhIGFBP-3 components and no increase in oxidation or aggregation level was observed with caffeine citrate, while admixtures of rhIGF-1/rhIGFBP-3 with ampicillin yielded lower mass recovery of rhIGF-1/rhIGFBP-3 components, which likely resulted from adduct formation. Furosemide was found to be physically incompatible with rhIGF-1/rhIGFBP-3. Our findings support the use of these methodologies for detection of protein modifications under various clinical administration conditions, and additionally supplement physical compatibility data studies of ultra-low concentrations of rhIGF-1/rhIGFBP-3 post co-administration to preterm infants with other medications (manuscript in-preparation).
