Bone Transport for Large Segmental Tibial Defects Using Taylor Spatial Frame versus the Ilizarov Circular Fixator.

OBJECTIVE: Bone transport has become the gold standard for treating large segmental tibial bone defects. The technique for application the Ilizarov circular fixator (ICF) has a long learning curve and is associated with many complications. There are few clinical studies on bone transport via the Taylor spatial frame (TSF). The main purpose of this study was to compare the radiological and clinical and outcomes of bone transport by using the TSF and the ICF. METHODS: There were 62 patients included in this retrospective study from June 2011 to June 2021 and distributed to two groups according to the fixation method: a TSF group consisting of 30 patients and an ICF group consisting of 32 patients. Demographic information, surgical duration, external fixation times, external fixation index, final radiographic results, complications, and clinical outcomes were recorded and examined. The clinical outcomes were assessed using the ASAMI criteria during the most recent clinical visit. Then, statistical analysis such as independent-samples t tests or chi-Square test was performed. RESULTS: The mean surgical duration in the TSF group was 93.8 ± 7.3 min, which was shorter than that in the ICF group (109.8 ± 1.4 min) (p < 0.05). Compared to the ICF group (10.2 ± 2.0 months), the TSF group (9.7 ± 1.8 months) had a shorter average external fixation time (p > 0.05). The external fixation index was 1.4 ± 0.2 m/cm and 1.5 ± 0.1 m/cm in the two groups. Moreover, there was no significant difference between the two groups. At the last follow-up visit, the medial proximal tibial angle (MPTA) and posterior proximal tibial angle (PPTA) in the TSF group were 88.1 ± 12.1° and 80.9 ± 1.3°, respectively. The MPTA and PPTA in the ICF group were 84.4 ± 2.4° and 76.2 ± 1.9°, respectively. There were statistically significant differences between the two groups (all p < 0.05). The complication rate was 50% in the TSF group and 75% in the ICF group. Moreover, the ASAMI score between the two groups was no statistically significant difference (p > 0.05). CONCLUSION: No statistically significant difference was found in clinical outcomes between the use of Taylor spatial frame and Ilizarov circular fixator for treating large segmental tibial bone defects. However, TSF is a shorter and simpler procedure that causes fewer complications and improves limb alignment.

In-Depth Characterization for Methionine Oxidization in Complementary Domain Region by Hydrophobic Interaction Chromatography.

The oxidation of the complementarity-determining region (CDR) in monoclonal antibodies (mAbs) is a critical quality attribute that can affect the clinical efficacy and safety of recombinant mAb therapeutics. In this study, a robust hydrophobic interaction chromatography (HIC) method was developed to quantify and characterize CDR oxidation variants in mAb-A by using a Proteomix Butyl-NP5 column. The HIC analysis revealed oxidation variants that eluted earlier than the main species with weaker hydrophobicity. It was found that Met105 in the CDR was more susceptible to oxidation. Additionally, it was noted that the oxidation of Met105 on a single heavy chain resulted in elution at a distinct position compared to the oxidation on two heavy chains. This observation led to the fractionation and enrichment of the oxidized variants for further evaluation of their biofunction. The study also demonstrated that the oxidation of Met105 did not impact the antigen-binding capacity but significantly reduced the PD-1/PD-L1 blockade activity of mAb-A. The HIC method, which was employed to quantify CDR oxidation, underwent validation and was subsequently utilized for stability studies as well as for assessing the similarity between mAb-A and its reference product.

Longitudinal changes in mitochondrial-associated measures and insulin resistance in youth with perinatally-acquired HIV in the U.S.

HIV infection and its treatment are associated with mitochondrial dysfunction and metabolic derangement. However, longitudinal changes in oxidative phosphorylation activities [Complex I (C1) and Complex IV (C4)], or venous lactate/pyruvate ratios (LPR), and their relationships with insulin resistance (IR), remain unclear in youth living with perinatally-acquired HIV (YPHIV). We measured venous LPR, C1, and C4 activities in blood cells and homeostatic model assessment for IR (HOMA-IR) over two years. Limited longitudinal differences in mitochondrial-related measures and IR were observed in YPHIV vs youth perinatally HIV-exposed but uninfected. There were no systematic differences in C1, C4, or HOMA-IR between the groups.

Characterization workflow for fragments detected in capillary electrophoresis sodium dodecyl sulfate analysis of therapeutic monoclonal antibodies.

Product-related fragments in monoclonal antibodies (mAbs) can have a significant impact on the efficacy and safety of the product. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is a commonly used method for fragment quantification, but it has challenges in peak identification due to the inability to enrich components and the incompatibility of SDS with mass spectrometry (MS). This article presents a workflow for identifying peaks in CE-SDS analysis. The workflow involves comparing the migration time of peaks with that of standards and utilizing MS analysis to identify fragments. By employing this innovative systematic workflow, we successfully identified the CE-SDS impurity peaks of seven antibody products. Among them, four products exhibited characteristic fragments associated with disulfide bonds (light chain [LC], heavy-light [HL] chain, heavy-heavy [HH] chain, and HH-LC) and a glycosylation-related fragment non-glycosylated heavy chain. Additionally, one product showed a fragment formed by the connection of HC_C130 and HC_C130, which is associated with a thioether bond. Furthermore, two other products displayed amino acid backbone breakage, with one product showing clipping at the HC region of A233-G285 and the other product showing clipping at the HC regions of A97-S158 and N342-T366. This workflow can be applied in early drug research, process development, or during the biologics license application stage to characterize fragments in therapeutic mAbs analyzed by CE-SDS.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing.

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins.

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

Identification of nonvolatile organic compounds (NVOCs) in biopharmaceuticals through non-target analysis and quantification using complexation-precipitation extraction.

Single-use systems in biopharmaceutical manufacturing can potentially release chemical constituents (leachables) into drug products. Prior to conducting toxicological risk assessments, it is crucial to establish the qualitative and quantitative methods for these leachables. In this study, we conducted a comprehensive screening and structure elucidation of 23 leachables (nonvolatile organic compounds, NVOCs) in two antibody drugs using multiple (self-built and public) databases and mass spectral simulation. We identified 7 compounds that have not been previously reported in medical or medicinal extractables and leachables. The confidence levels for identified compounds were classified based on analytical standards, literature references, and fragment assignments. Most of the identified leachables were found to be plasticizers, antioxidants, slip agents or polymer degradants. Polysorbate (namely Tween) is commonly used as an excipient for protein stabilization in biopharmaceutical formulations, but its ionization in liquid chromatography-electrospray ionization mass spectrometry can interfere with compound quantification. To address this, we employed a complexation-precipitation extraction method to reduce polysorbate content and quantify the analytes. The developed quantitative method for target NVOCs demonstrated high sensitivity (limit of quantification: 20 or 50 µg/L), accuracy (recoveries: 77.2 to 109.5 %) and precision (RSD ≤ 8.2 %). Overall, this established method will facilitate the evaluation of NVOC safety in drug products.

The sources, properties, extraction, biosynthesis, pharmacology, and application of lycopene.

Lycopene is an important pigment with an alkene skeleton from Lycopersicon esculentum, which is also obtained from some red fruits and vegetables. Lycopene is used in the food field with rich functions and serves in the medical field with multiple clinical values because it has dual functions of both medicine and food. It was found that lycopene was mainly isolated by solvent extraction, ultrasonic-assisted extraction, supercritical fluid extraction, high-intensity pulsed electric field-assisted extraction, enzymatic-assisted extraction, and microwave-assisted extraction. Meanwhile, it was also obtained via 2 synthetic pathways: chemical synthesis and biosynthesis. Pharmacological studies revealed that lycopene has anti-oxidant, hypolipidemic, anti-cancer, immunity-enhancing, hepatoprotective, hypoglycemic, cardiovascular-protective, anti-inflammatory, neuroprotective, and osteoporosis-inhibiting effects. The application of lycopene mainly includes food processing, animal breeding, and medical cosmetology fields. It is hoped that this review will provide some useful information and guidance for future study and exploitation of lycopene.

A Novel Method for Monitoring N-Nitrosamines Impurities Using NH2-MIL-101(Fe) Mediated Dispersive Micro-Solid Phase Extraction Coupled with LC-MS/MS in Biopharmaceuticals.

A highly efficient and convenient method for the simultaneous determination of 12 N-nitrosamines (NAs) has been developed using an amine-functionalized metal-organic framework (NH2-MIL-101(Fe)) as sorbent for dispersive micro-solid phase extraction (D-µSPE) coupled with LC-MS/MS in biopharmaceuticals. The experimental variables involved in the extraction process (i.e., amount of the sorbent, extraction time, desorption time, ionic strength, desorption solvent and volume) were optimized to achieve the best extraction efficiency of the target analytes. Under the optimum conditions, the method was successfully validated, showing good linearity in the range of 0.5-3.0 µg/L with determination coefficients (R2) higher than 0.990, repeatability (RSD ≤ 10.0%, spiked level at 2.0 µg/L) and precision (RSD ≤ 8.2%). The limit of detection (LOD) and limit of quantitation (LOQ) were in the range of 0.005-0.025 µg/L and 0.010-0.250 µg/L, respectively. Satisfactory recoveries ranging from 82.4 to 116.8% were obtained by spiking standards at three different concentrations (0.5 µg/L, 2.0 µg/L and 3.0 µg/L). Other validation parameters, including specificity, stability, and robustness, met the validation criteria. More importantly, the plausible adsorption mechanism on NH2-MIL-101(Fe) was proposed by Fourier-transform infrared (FTIR) spectra technique. Finally, this method was successfully applied to detect trace nitrosamines in biopharmaceuticals.

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry.

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1'­carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SIGNIFICANCE: This article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.