Development of Protein-Specific Analytical Methodologies to Evaluate Compatibility of Recombinant Human (rh)IGF-1/rhIGFBP-3 with Intravenous Medications Co-Administered to Neonates.

The protein complex of recombinant human insulin-like growth factor-1 and insulin­like growth factor binding protein­3 (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).

Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles.

This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product.

Characterization of small protein aggregates and oligomers using size exclusion chromatography with online detection by native electrospray ionization mass spectrometry.

Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale.

Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD.

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

Studies of pH-dependent self-association of a recombinant form of arylsulfatase A with electrospray ionization mass spectrometry and size-exclusion chromatography.

Arylsulfatase A is an endogenous enzyme that is responsible for the catabolism and control of sulfatides in humans. Its deficiency results in the accumulation of sulfatides in the cells of the central and peripheral nervous system leading to the destruction of the myelin sheath and resulting in metachromatic leukodystrophy (MLD), a neurodegenerative lysosomal storage disease. A recombinant human form of this glycoprotein (rhASA) is currently under development as an enzyme replacement therapy. At neutral and alkaline pH, this protein exists as a homodimer but converts to an octameric state in the mildly acidic environment of the lysosome, and a failure to form an octamer results in suboptimal catalytic activity (most likely due to a diminished protection from lysosomal proteases). Despite the obvious importance of the rhASA oligomerization process, its mechanistic details remain poorly understood. In this work, we use size exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI MS) to monitor the dimer-to-octamer transition as a function of both solution pH and protein concentration. While SEC clearly shows different profiles (i.e., retention time differences) for rhASA when the chromatography is performed at neutral and lysosomal pH, consistent with changing oligomerization states, no resolved peaks could be observed for either octamer or dimer when analyzed at intermediate pH (5.5-6.5). This could be interpreted either as the result of a rapid dimer-to-octamer interconversion on the chromatographic time scale or as a consequence of the presence of previously unidentified intermediate species (e.g., tetramer and/or hexamer). In contrast, ESI MS provides strong evidence of the dimer-to-octamer transition state that occurs when the analysis is performed within a narrow pH range (6.0-7.0). Octamer assembly was shown to be a highly cooperative process with no intermediate states that are populated to detectable levels. A tetrameric state of rhASA exists at equilibrium with a dimer at neutral pH but does not appear to be involved in the octamer assembly process.

Impact of oxidation on protein therapeutics: conformational dynamics of intact and oxidized acid-ß-glucocerebrosidase at near-physiological pH.

The solution dynamics of an enzyme acid-ß-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40-60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking.

Characterization of gene-activated human acid-beta-glucosidase: crystal structure, glycan composition, and internalization into macrophages.

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-beta-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K(m) and V(max)) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Infratentorial angioleiomyoma: a new location for a rare neoplastic entity.

Angioleiomyomas are benign neoplasms most often located in the subcutaneous tissue of middle-aged individuals and usually confined to the subcuticular and deep dermal layers of the lower extremities. An intracranial site for this tumor is exceedingly rare, with very few reports documenting locations in the neuraxis. To the authors' knowledge the present case represents the first reported instance of an infratentorial angioleiomyoma. The authors conducted a review of selected English-language papers published since 1960 describing well-documented cases of intracranial vascular leiomyomas, with detailed information on the clinical presentation, radiology, pathology, and particulars of surgical management in each case.

Angiosarcoma of the of scalp with calvarium involvement in a 50-year-old African-American man.

BACKGROUND: Angiosarcoma of the scalp is a rare, aggressive, and deadly cancer that affects mainly elderly Caucasian men. OBJECTIVES: The insidious and masquerading presentation of angiosarcoma poses enormous diagnostic challenges for primary care providers. PATIENTS/METHODS: We present a case of a 50-year-old black man referred for evaluation of a 3.7-cm-x-5.4-cm ulcerated, fluctuant scalp lesion that had failed to respond to different antibiotics and proper wound care. RESULTS: Surgical excision and subsequent histopathology revealed angiosarcoma. CONCLUSIONS: This case report highlights the importance of high index of suspicion for early diagnosis of cancerous lesions in wounds and stresses the need to include angiosarcoma in the differential diagnosis for all face and scalp lesions, as early detection may save lives. A comprehensive literature review is also presented.