A Genetically Engineered "Reinforced Concrete" Scaffold Regulates the N2 Neutrophil Innate Immune Cascade to Repair Bone Defects.

The innate immune response is crucial to inflammation, but how neutrophils and macrophages act in bone repair and tissue engineering treatment strategies await clarification. In this study, it is found that N2 neutrophils release stronger "eat me" signals to induce macrophage phagocytosis and polarize into the M2 anti-inflammatory phenotype. Guided by this biological mechanism, a mesoporous bioactive glass scaffold (MBG) is filled with hyaluronic acid methacryloyl (HAMA) hydrogel loaded with Transforming growth factor-ß1 (TGFß1) adenovirus (Ad@H), constructing a genetically engineered composite scaffold (Ad@H/M). The scaffold not only has good hydrophilicity and biocompatibility, but also provides mechanical stress support for bone repair. Adenovirus infection quickly induces N2 neutrophils, upregulating NF-κB and MAPK signaling pathways through Toll-like receptor 4 (TLR4) to promote the inflammatory response and macrophage phagocytosis. Macrophages perform phagocytosis and polarize towards the M2 phenotype, mediating the inflammatory response by inhibiting the PI3K-AKT-NF-κB pathway, maintaining homeostasis of the osteogenic microenvironment. The role of the Ad@H/M scaffold in regulating early inflammation and promoting long-term bone regeneration is further validated in vivo. In brief, this study focuses on the cascade of reactions between neutrophils and macrophage subtypes, and reports a composite scaffold that coordinates the innate immune response to promote bone repair.

TRUST Technique for Neurointervention: A Promising Alternative for Complex Cases.

BACKGROUND: Neurointervention via Transradial Access (TRA) is becoming increasingly popular as experience with this technique increases. However, approximately 8.6-10.3% of complex TRA cases are converted to femoral access due to a lack of support or radial artery spasm. This study aimed to assess the efficacy and safety of the TRUST (trans-radial coaxial catheter technique using a short sheath, Simmons catheter, and Tethys intermediate catheter) technique in interventional procedures via TRA. METHODS: This was a single-center retrospective analysis of 16 patients admitted to our institute between January 2023 to May 2023 to undergo endovascular interventions with the TRUST technique via the TRA. RESULTS: The mean age of the study population was 63.8 years, and 62.5% were male (10/16). The most common procedure was intracranial atherosclerotic stenosis (93.75%, 15/16). All procedures were performed successfully, and the most common procedures in our cohort were ballooning (50.0%, 8/16), stenting (18.75%, 3/16), and both procedures combined (31.25%, 1/16). All procedures were performed using the TRA, and the distal and proximal radial arteries were used for access in 31.35% (5/16) and 68.75% (11/16) of the cases, respectively. Technical success was achieved in all patients and most cases demonstrated mTICI ≥2b recanalization (93.75%, 15/16). In this case, no major access-site complications occurred. CONCLUSION: The TRUST technique is technically safe and feasible and had a high technical success rate and low complication rate in our study. These results demonstrate that the TRUST technique is a promising alternative for patients undergoing complex neurointerventions.

Ligand-Selective Targeting of Macrophage Hydrogel Elicits Bone Immune-Stem Cell Endogenous Self-Healing Program to Promote Bone Regeneration.

Targeting macrophages can facilitate the site-specific repair of critical bone defects. Herein, a composite hydrogel, gelatin-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogel (GBMgel), is constructed via the self-assembly of mesoporous bioactive glass on polysaccharide structures, through the Schiff base reaction. GBMgel can efficiently capture macrophages and drive the recruitment of seed stem cells and vascular budding required for regeneration in the early stages of bone injury, and the observed sustained release of inorganic silicon ions further enhances bone matrix deposition, mineralization, and vascular maturation. Moreover, the use of macrophage-depleted rat calvarial defect models further confirms that GBMgel, with ligand-selective macrophage targeting, increases the bone regeneration area and the proportion of mature bone. Mechanistic studies reveal that GBMgel upregulates the TLR4/NF-κB and MAPK macrophage pathways in the early stages and the JAK/STAT3 pathway in the later stages; thus initiating macrophage polarization at different time points. In conclusion, this study is based on the endogenous self-healing properties of bone macrophages, which enhances stem cell homing, and provides a research and theoretical basis upon which bone tissue can be reshaped and regenerated using the body's immune power, providing a new strategy for the treatment of critical bone defects.

Ligand-Screened Cerium-Based MOF Microcapsules Promote Nerve Regeneration via Mitochondrial Energy Supply.

Although mitochondria are crucial for recovery after spinal cord injury (SCI), therapeutic strategies to modulate mitochondrial metabolic energy to coordinate the immune response and nerve regeneration are lacking. Here, a ligand-screened cerium-based metal-organic framework (MOF) with better ROS scavenging and drug-loading abilities is encapsulated with polydopamine after loading creatine to obtain microcapsules (Cr/Ce@PDA nanoparticles), which reverse the energy deficits in both macrophages and neuronal cells by combining ROS scavenging and energy supplementation. It reprogrames inflammatory macrophages to the proregenerative phenotype via the succinate/HIF-1α/IL-1ß signaling axis. It also promotes the regeneration and differentiation of neural cells by activating the mTOR pathway and paracrine function of macrophages. In vivo experiments further confirm the effect of the microcapsules in regulating early ROS-inflammation positive-feedback chain reactions and continuously promoting nerve regeneration. This study provides a new strategy for correcting mitochondrial energy deficiency in the immune response and nerve regeneration following SCI.

Angiographic Pattern of M1 Stenosis Predicts Territorial Stroke in Patients Receiving Aggressive Medication without Stenting.

OBJECTIVE: To evaluate the relationship between different angiographic patterns of middle cerebral artery M1 segment stenosis and related territorial stroke in patients receiving aggressive medical treatment without stenting. METHODS: We retrospectively reviewed our patient registry database to identify ICAS patients diagnosed by digital subtraction angiography between January 2017 and December 2020 and identified 3 different angiographic patterns (normal, shift, and dilation) in 124 patients with M1 stenosis. The association between these patterns and recurrent ischemic stroke in the M1 territory was analyzed. RESULTS: The rates of recurrent M1 territorial stroke and transient ischemic attack in the normal group, shift group, dilation group and shift-dilation group were 34.5%, 35.0%, 78.3%, and 44.4% respectively. In patients with the shift pattern, the rate of recurrent stroke is significantly higher at a deflection angle ≥9.32° than at a deflection angle <9.32°(P < 0.05). In patients with dilation pattern, the rate of recurrent stroke is significantly higher than patients with non-dilation pattern (72.3% vs. 36.8%, P < 0.05). CONCLUSIONS: Angiographic patterns of M1 stenosis may predict recurrent territorial strokes, thus providing a surrogate marker to identify high-risk patients for potential endovascular treatment.

The impact of thrombus surface morphology of the basilar artery on the successful rate of mechanical thrombectomy.

Objective: The present study aimed to investigate the impact of angiographic thrombus surface morphology on the angiographic and clinical outcomes of basilar artery occlusion (BAO). Methods: This retrospective study included 141 patients with acute BAO who underwent mechanical thrombectomy (MT). We categorized thrombus surface phenotypes as either regular (smooth and straight, either convex or concave) or irregular. Patients with BAO were grouped based on the presence of a regular or irregular phenotype, and we compared their angiographic and clinical outcomes. Results: In total, 52.5% (74/141) of acute BAO patients exhibited a regular thrombus morphology. These patients had a higher rate of first-pass effect (28.4% vs. 4.5%, p = 0.0002) and fewer retrieval attempts (2 vs. 2; p = 0.0198) compared to those with irregular morphology. Among patients treated with contact aspiration (CA), the regular thrombus morphology showed a higher first-pass success rate (45.7% vs. 12.8%; p = 0.0017), a shorter procedural duration (46 vs. 50 min; p = 0.0159), and fewer retrieval attempts (1 vs. 2; p = 0.0338) compared to stent retriever (SR) thrombectomy. Both the regular thrombus morphology (OR 7.72, 95% CI 2.02-29.52; p = 0.003) and using CA as the first-line treatment (OR 3.37, 95% CI 1.12-10.13; p = 0.031) independently predicted first-pass success. Conclusion: For BAO patients treated with CA as the primary strategy, the presence of a regular thrombus surface might predict higher first-pass success and shorter procedural duration. A diligent assessment of thrombus morphology within the MT workflow could improve the feasibility of procedural techniques.

Revisiting the predictive role of truncal-type occlusion sign for acute large vessel stroke treated by endovascular thrombectomy: A scoping review.

Background and purpose The truncal type occlusion (TTO) sign observed during endovascular thrombectomy (EVT) is thought to predict the etiology and prognosis of acute ischemic stroke (AIS) due to large vessel occlusion (LVO). However, the interpretation of the present results and the clinical utility of this sign needs further investigation. This scoping meta-review aimed to assess the predictive value of the TTO sign, thus identifying methodological limitations in current study designs. Methods Studies published up to January 2023 were identified by systematically searching PubMed, Embase, and Web of Science. A meta-analysis was performed to quantitatively synthesize the evidence on the predictive value of the TTO sign. An 8-point scale was introduced to narratively summarize the current evidence level and methodological quality of included studies. Results We included 10 studies in this review. For the prediction of intracranial atherosclerotic stenosis, the sensitivity, specificity, PLR and NLR of the TTO sign were 0.73, 0.87, 5.5 and 0.31, respectively (all p<0.05). For recanalization failure after primary thrombectomy, the sensitivity, specificity, PLR and NLR were 0.44, 0.91, 4.9 and 0.61, respectively (all p<0.05). The strength of evidence was low due to the methodological limitations and lack of adjustment for potential confounders. Conclusion The predictive values of the TTO sign for the etiology of LVO-AIS was considerable but seemed limited for current interpretation. Several confounders could influence the determination and predictive value of the TTO sign, requiring methodological adjustments in future research. Endovascular practitioners encountering this sign during thrombectomy should draw specific attention to stroke etiology, thus promoting timely adjustment of intra- and postprocedural strategies.

Initial clinical experience with Xpert-Pro peripheral self-expanding stent system for internal carotid artery dissection: Two case reports.

Background: The standard treatment for internal carotid artery (ICA) dissection is obscure. Current therapeutic strategies include the use of antiplatelet drugs, anticoagulant drugs, intravenous thrombolysis, and endovascular treatment. Endovascular treatment is important in acute internal carotid artery dissection. This study reports two acute internal carotid artery dissection cases that were treated successfully using the Xpert-Pro peripheral self-expanding stent system. Case summary: The first case was of a 38-year-old male patient with transient speechlessness and paralysis of the right limb in July 2021. Cervical computed tomographic angiography (CTA) showed ICA occlusion on the left side. Digital subtraction angiography (DSA) showed severe stenosis of the C1 segment of the left internal carotid artery with intermural hematoma. The patient subsequently underwent Xpert-Pro peripheral self-expanding stent implantation, and his condition stabilized. The second case was of a 56-year-old male patient with speechlessness and paralysis of the right limb. Cervical CTA showed a dissected left ICA, and DSA showed an occluded left ICA and middle cerebral artery. The patient subsequently underwent stent implantation, and his condition stabilized.

Branch-site occlusion sign predicts the embolic origin of acute ischemic stroke: a meta-analysis.

Objective: The study aimed to investigate whether branch-site occlusion (BSO) sign could predict the etiology of acute intracranial large artery occlusion (ILVO) and the stentriever (SR) response. Methods: We systematically reviewed studies that evaluated the predictive role of BSO for the etiology of ILVO-AIS or EVT outcome between 1 January 2000 and 31 August 2022 from PubMed, Embase, and Web of Science. Results: The sensitivity and specificity of BSO sign predicting etiology of ILVO-AIS were 0.87 (95% CI 0.81-0.91) and 0.64 (95% CI 0.33-0.87), respectively. The sensitivity and specificity of BSO sign predicting stentriever response were 0.84 (95% CI 0.63-0.94) and 0.61 (95% CI 0.18-0.92), respectively. Conclusion: The BSO sign could be a valid and precise imaging marker to predict embolism caused ILVO-AIS and recanalization success by SR without rescue therapy.

Comparison of three common shoulder injections for rotator cuff tears: a systematic review and network meta-analysis.

OBJECTIVE: To compare the clinical effectiveness of three common shoulder injections mentioned in the guidelines [corticosteroid, sodium hyaluronate (SH) and platelet-rich plasma (PRP)] on rotator cuff tears. MATERIAL AND METHODS: The PubMed, Embase and Cochrane Library databases were systematically searched up to June 1, 2022, for randomized controlled trials (RCTs) and prospective studies on the three injection therapies for rotator cuff tears. The main results were pain relief and functional improvement at 1-5 months and over 6 months, pooled using a network meta-analysis and ranked by SUCRA score. The risk of bias of the included studies was assessed using the Cochrane Collaboration tool. RESULTS: Twelve RCTs and 4 prospective studies comprising a total of 1115 patients were included in the review. Three prospective studies were judged to be at high risk of selection bias and performance bias, and one was considered as having a high risk of detection bias. SH injection ranked first in the short term in pain relief (MD: - 2.80; 95%CI - 3.91, - 1.68) and functional improvement (MD:19.17; 95%CI 12.29, 26.05), while PRP injection obtained better results in the long term in both pain relief (MD: - 4.50; 95%CI - 4.97, - 4.03) and functional improvement (MD:11.11; 95%CI 0.53,21.68). CONCLUSIONS: PRP injection has the potential to successfully treat rotator cuff tears as an alternative to corticosteroids in the long term, in terms of either therapeutic efficiency or adverse effects, followed by SH injection. More research is needed to make high-quality recommendations on treatment options for injection treatments of rotator cuff tears.

Staged angioplasty: A sensible approach to prevent hyperperfusion syndrome after carotid artery stenting? A meta-analysis.

OBJECTIVE: To investigate whether staged angioplasty (SAP) is a safe and effective treatment to prevent hyperperfusion syndrome after carotid artery stenting (CAS). METHODS: A systematic literature search was performed according to established criteria to identify eligible articles published before October 2020. Pooled dichotomous data were presented as odds ratios (OR) and corresponding 95% confidence intervals (CI) using random-effect models. The efficacy endpoints were hyperperfusion syndrome (HPS), hyperperfusion phenomenon (HPP), and intracerebral hemorrhage (ICH). The safety endpoint was post-procedural thromboembolic events. The feasibility of the procedure was assessed by device-related adverse events (vessel dissection and failed angioplasty) in SAP. RESULTS: Ten studies (1030 participants) were eligible. SAP was superior to regular CAS in preventing HPS (OR = 0.35, 95% CI 0.14-0.86, P = 0.02). There was no significant difference in the rate of thromboembolic events between the SAP group and the regular CAS group. The rates of vessel dissection and failed angioplasty with the use of a 3.0-mm-diameter balloon were 5.4% and 0.4%, respectively. CONCLUSION: SAP may reduce the incidence of post-CAS HPS without increasing procedure-related complications. A 3.0-mm-diameter balloon used in SAP may be appropriate for Asian populations. However, the confounded study design and confused definitions of reporting items hinder the current recommendation of SAP in clinical use.

Does radiological conjugate eye deviation sign play a role in acute stroke imaging? A meta-analysis.

BACKGROUND AND PURPOSE: The diagnostic value of non-contrast CT (NCCT) in acute stroke imaging remains indispensable, especially under emergency conditions with limited resources. The radiological conjugate eye deviation (RCED) has been demonstrated as a NCCT sign to predict acute ischemic stroke (AIS) or AIS secondary to large vessel occlusion (LVO) in recent studies. We performed a meta-analysis to gain a better understanding into the predictive role of RCED for AISs and LVO-AISs. METHODS: We conducted a systematic literature search using PubMed, Embase, and Cochrane. The search focused on studies published between January 2000 and August 2020 that reported the predictive value of RCED for the diagnosis of AIS or LVO-AIS. Principal measurements of the meta-analysis were the overall sensitivity, specificity, the positive likelihood ratio (PLR), and the negative likelihood ratio (NLR) of RCED in predicting AIS and LVO-AIS. RESULTS: We included 11 studies (n = 2304). For AIS, RCED had a sensitivity of 0.37 (95% CI 0.27-0.47), a specificity of 0.86 (95% CI 0.73-0.93), the area under the receiver operating characteristic curve (AUC) was 0.58 (95% CI 0.53-0.62), PLR was 2.5 (95% CI 1.5-4.4), and NLR was 0.74 (95% CI 0.65-0.84). For LVO-AIS, RCED had a sensitivity of 0.63 (95% CI 0.46-0.77), a specificity of 0.77 (95% CI 0.71-0.82), AUC was 0.63 (95% CI 0.46-0.77), PLR was 2.7 (95% CI 1.7-4.3), and NLR was 0.49 (95% CI 0.3-0.78). CONCLUSION: RCED can be used to predict LVO-AIS. It is expected that this method will be extensively used and validated in acute stroke imaging, especially under emergency conditions with limited resources.

Better late than never: initial experience of intra-arterial pulsed-urokinase-injection as a salvage therapy for refractory sudden sensorineural hearing loss.

BACKGROUND AND PURPOSE: Cochlear vascular micro-thrombosis has been hypothesized as one of the pathogenic mechanisms for sudden sensorineural hearing loss (SSNHL) refractory to regular management. This study aimed to evaluate the feasibility and safety of intra-arterial pulsed-injection urokinase (IAPU) as a salvage therapy for SSNHL after the failure of conventional therapy. METHODS: We retrospectively reviewed our patient database to identify refractory SSNHL patients between November 2017 and July 2020. Study outcomes before and after the IAPU therapy were compared between IAPU and conventional therapy groups. RESULTS: Sixty-seven moderate-profound SSNHL patients (29 in IAUP group, 38 in control group) were included in this study. Compared to the control group, patients in the IAPU group showed more significant improvement in pure tone average (PTA) (34.2 ± 23.5 vs. 10.7 ± 13.1, p < 0.001) and degree of hearing recovery (total: 20.7% vs. 5.3%, partial: 24.1% vs. 10.5%, mild: 27.6% vs. 13.2% and non: 27.6% vs. 71.1%) 2 weeks after admission. In the IAPU group, a significant improvement of PTA (86.6 ± 11.5 vs. 54.6 ± 20.1 dB, p < 0.005) was observed on the first day after IAPU treatment. CONCLUSION: In carefully selected SSNHL cases with a highly suspected vascular origin, IAPU is a safe and effective therapy when conventional treatments have failed. Despite the encouraging findings of our work, large studies are needed to better investigate the strengths and limitations of this salvage therapy.

Cognition effectiveness of continuous positive airway pressure treatment in obstructive sleep apnea syndrome patients with cognitive impairment: a meta-analysis.

Obstructive sleep apnea (OSA) is a common respiratory disorder characterized by recurrent pharyngeal collapses during sleep leading to intermittent hypoxia and sleep disruption. Cognitive challenges and high risks of cognitive impairment, including Alzheimer's disease (AD), are closely associated with OSA. Currently, continuous positive airway pressure (CPAP) is widely used in the treatment of OSA. However, whether CPAP benefits cognitive functions in patients with OSA remains elusive. Here, we identified published studies through a systematic review of PubMed, Cochrane Library, Embase, Wanfang Data, CBM, and CNKI from January 1, 1970, to July 1, 2020. 288 patients from 7 articles (one was excluded in the meta-analysis for it was a follow-up study) were included in the present study. It revealed that cognitive functions of OSA patients with mild cognitive impairment (MCI) or AD were mildly but significantly improved after CPAP treatment (SMD 0.49, 95% CI 0.11-0.86), especially long-term CPAP treatment (SMD 0.56, 95% CI 0.10-1.02, p = 0.02), as measured by Mini-Mental State Examination (MMSE) (SMD 0.49, 95%CI 0.11-0.86). However, no significant cognition benefits were detected by the Montreal Cognitive Assessment (SMD 0.43, 95% CI 0.85-1.72). In terms of heterogeneity, cognitive improvements by CPAP were detectable on OSA patients either at a younger age or over longer periods of CPAP treatment. Therefore, our findings highlight the partial efficiency of CPAP treatment in cognition improvement of OSA patients with MCI or AD.

Two-Factor-Based Public Data Protection Scheme in Smart Ocean Management.

Nowadays, two-factor data security protection has become a research hotspot in smart ocean management. With the increasing popularity of smart ocean management, how to achieve the two-factor protection of public data resources in smart ocean management is a serious problem to be tackled. Furthermore, how to achieve both security and revocation is also a challenge for two-factor protection. In this paper, we propose a two-factor-based protection scheme with factor revocation in smart ocean management. The proposed scheme allows data owners (DOs) to send encrypted messages to users through a shipboard server (SS). The DOs are required to formulate access policy and perform attribute-based encryption on messages. In order to decrypt, the users need to possess two factors. The first factor is the user's secret key. The second factor is security equipment, which is a sensor card in smart ocean system. The ciphertext can be decrypted if and only if the user gathers the key and the security equipment at the same time. What is more, once the security equipment is lost, the equipment can be revoked and a new one is redistributed to the users. The theoretical analysis and experiment results indeed indicate the security, efficiency, and practicality of our scheme.

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.

Assessment of stable isotope incorporation into recombinant proteins.

Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture.

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system.

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.

Analysis and characterization of aggregation of a therapeutic Fc-fusion protein.

Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at -30 °C, even though the protein was stable at 4 and -80 °C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC-MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein.