Timed up and go demonstrates strong interrater agreement and criterion validity as a functional test in geriatric dogs.

OBJECTIVE: To measure interobserver agreement for 4 functional tasks and their summed geriatric functional score (GFS) and correlate tasks and GFS with client-specific outcome measurements (CSOMs): Canine Brief Pain Inventory (CBPI) pain severity, CBPI pain interference, and Liverpool Osteoarthritis in Dogs. ANIMALS: 89 geriatric dogs were recruited between April and September 2023 from staff, friends, and clients of the Cornell University College of Veterinary Medicine with a median age of 11.0 years and weight of 26.4 kg. METHODS: Dogs underwent 4 sequential functional tests: timed up and go (TUG), cavallettis, figure 8s, and down to stands. Two observers independently scored each dog. The GFS was calculated based on the summed scores of the individual tests. Additional information collected included signalment, weight, measurements reflecting the comorbidities of aging (body condition score and muscle condition score), and CSOMs. RESULTS: Strong interrater agreement was found for all functional tests. The TUG in seconds (sTUG) and figure 8s demonstrated significant (P < .05) moderate to strong correlations to all CSOMs. The GFS showed similar significant correlations with all CSOMs except CBPI pain severity; however, when correlating individual tests to CSOMs, only figure 8s and TUG were significantly contributing to GFS results. Receiver operating characteristic curve analysis defined highly functional dogs as those completing the sTUG in under 3.83 seconds. The sTUG represented the best test for geriatric function given it was objective, reliable, correlated well to CSOMs, and could help identify highly functioning dogs. CLINICAL RELEVANCE: The sTUG appears to be the first practical and reliable functional test of canine geriatric mobility.

Review of cellular therapies provides new insights into the potential treatment of diverse neurologic diseases in horses and dogs.

Neurological diseases and injuries in veterinary patients (horses, dogs, and cats) are complex, and effective treatment options are limited. Neuronal loss, damage to nerve conduction pathways, and inflammation and scarring associated with spinal cord injury pose major challenges in managing many neurological diseases. Furthermore, most of these neuropathologies lack definitive pharmacological treatments, driving interest and research into novel interventions. Our objective is to provide a narrative review of the current literature surrounding cellular therapies including neuronal and glial stem cells, neurotrophic factors, mesenchymal stem or stromal cells, and cells derived from induced pluripotent stem cells for the treatment of diverse neurological pathologies. Cellular therapies have the potential for cellular replacement, immune modulation, and paracrine signaling and the flexibility of being used alone or alongside surgical intervention. Mesenchymal stem or stromal cells are arguably the most researched cellular therapy and have been administered intrathecally, IV, intra-arterially, intranasally, and intraspinally with few adverse reactions. Limited clinical and experimental studies have suggested efficacy in diseases including acute spinal cord injury and intervertebral disc disease. Little is currently known about the safety and efficacy of neural stem cells, precursor cell administration, and induced pluripotent stem cell-derived treatments. Further research is necessary to determine the efficacy and long-term safety of cellular therapies. Future aims should include larger controlled clinical trials in companion animals for common neurologic conditions including acute spinal cord injury, intervertebral disc disease, peripheral nerve injury, degenerative neuropathies, and age-associated cognitive decline.

Literature review details and supports the application of platelet-rich plasma products in canine medicine, particularly as an orthobiologic agent for osteoarthritis.

Platelet-rich plasma (PRP) and other platelet-derived products represent a subset of regenerative medicine and have been researched in the veterinary community for the treatment of osteoarthritis, soft tissue wounds, tendinopathies, periodontitis, and fracture repairs. PRP is simple to produce, relatively affordable, safe, and can be delivered on site, making it an appealing therapeutic agent in veterinary medicine. As an orthobiologic for the treatment of osteoarthritis, it is one of few interventions with clinical study support that possess anabolic potential. Platelet product variability is wide ranging and often described in terms of cellular content or platelet enrichment. Growth factors associated with platelet activation and subsequent degranulation may mediate inflammation, modulate cellular immune response, and promote tissue repair. Product composition, dosage, and application likely influence treatment outcomes depending on the classification of the disease targeted. Sufficient canine data regarding the formulation and clinical application of canine PRP exist to warrant review. The aim of this narrative is to provide scientific background and clinical insight for veterinarians regarding platelet product content/formulation, mechanisms of action, considerations for use, and clinical application in dogs.

Interactions Between Biologic Therapies and Other Treatment Modalities.

Biologic therapies are becoming increasingly utilized by veterinarians. The literature regarding the interaction of biologic therapies with other therapeutics is still in its infancy. Initial studies have examined the effects of exercise, stress, various pharmaceutical interventions, extracorporeal shockwave, therapeutic laser, and hyperbaric oxygen on biologic therapies. Continued research is imperative as owners and veterinarians increasingly choose a multimodal approach to injury and illness. Further, understanding the effects of concurrently administered treatments and pharmaceuticals as well as the health status of the horse is imperative to providing the optimal therapeutic outcome.

Retrospective analysis describes safety of therapeutic joint injections in dogs.

OBJECTIVE: To retrospectively investigate the safety of canine therapeutic IA injections, describing and correlating adverse events with the number of injections per visit, joint injected, signalment, body condition score, type, and volume of injectate. SAMPLE: There were 505 joint injections across 283 visits for 178 client-owned dogs, including the shoulder, elbow, carpus, hip, stifle, tarsus, and metacarpophalangeal. PROCEDURES: A search was performed of the Cornell University Hospital for Animals medical records for relevant data, identifying dogs treated with therapeutic joint injections and rechecked between 2010 and 2022. RESULTS: Minor complications were noted in 70 of 283 visits and included transient soreness (18.4%, lasting a median of 2 days; range, 1 to 20 days) and gastroenteritis (6.8%). One case of septic arthritis (1/505 joints), which possessed risks of a hematogenous source, was the only potential major complication. Soreness was not correlated with the number of joints injected per visit. Larger volumes of injectate normalized to body size were more likely to be associated with transient soreness in the stifle and tarsus. Across injectates, only stem cells had significantly increased odds of soreness. Gastroenteritis was not associated with the type of injectate. CLINICAL RELEVANCE: Therapeutic joint injections in dogs are safe, with an extremely low risk of major adverse effects. Transient soreness is a commonly expected minor adverse event. The use of stem cells or larger injectate volumes (confined to the stifle and smaller distal joints) may be more likely to invoke discomfort.

Serum myostatin decreases in exercising and aging Alaskan sled dogs, while growth and differentiation factor 15 remains unaltered.

OBJECTIVES: To evaluate the serum concentrations of myostatin and growth and differentiation factor 15 (GDF-15) in Alaskan Husky sled dogs participating in a 350-mile (560-km) race and in an older population, and to examine correlations between changes in serum concentrations and body condition scores (BCSs). ANIMALS: Dogs were recruited from 3 teams of Alaskan Huskies participating in the Alaskan-Yukon Quest sled-dog race and retirees from a research sled-dog colony. PROCEDURES: Serum samples and BCSs were collected prior to racing, midway, and postrace; and in an older cohort (13 to 14 years). Myostatin and GDF-15 concentrations were assessed using commercially available ELISA kits. RESULTS: The median myostatin prerace concentration (9,519 pg/mL) was significantly greater than the mid- and postrace concentrations (7,709 pg/mL and 3,247 pg/mL, respectively). The prerace concentration was also significantly greater than that of the retired sled group dogs at 6,134 pg/mL. GDF-15 median serum concentrations did not change significantly across any racing time point (approx 350 pg/mL) or in the older cohort. No significant correlations were observed between changes in BCS and myostatin or GDF-15 concentrations. CLINICAL RELEVANCE: Serum myostatin decreases dramatically, yet no correlations to loss of BCS could be found. Myostatin signaling may be involved in maintaining hypertrophic signaling during intense exercise. Neither racing distance nor geriatric/retirement status appears to have an effect on serum GDF-15 concentration. Myostatin was less in the older, retired sled dogs compared to the younger racing cohort. Such differences highlight the roles that fitness level and age play regarding myostatin levels.

Canine Geriatric Rehabilitation: Considerations and Strategies for Assessment, Functional Scoring, and Follow Up.

Geriatric animals account for half of the pet population in the United States with their numbers increasing annually. Furthermore, a significant percentage of veterinary patients with movement limitations could be grossly categorized as geriatric and living within the end stage of their predicted lifespans. Because mobility is correlated to quality of life and time to death in aging dogs, a major goal in optimizing canine geriatric health is to improve functional movement. Within the geriatric population, identifying disabilities that affect daily living and quality of life may be used by the rehabilitation practitioner to provide stronger prognoses, treatment goals, and outcome measures. Examples of such means are described within this review. In human medicine, the concept of "optimal aging", or "healthy aging", has emerged in which inevitable detrimental age-related changes can be minimized or avoided at various levels of physical, mental, emotional, and social health. Both environment and genetics may influence aging. Identifying and improving environmental variables we can control remain a key component in optimizing aging. Furthermore, diagnosing and treating age related comorbidities common to older populations allows for improved quality of life and is often directly or indirectly affecting mobility. Obesity, sarcopenia, and a sedentary lifestyle are a trifecta of age-related morbidity common to both people and dogs. Healthy lifestyle choices including good nutrition and targeted exercise play key roles in reducing this morbidity and improving aging. Disablement models act as essential tools for creating more effective physiotherapy plans in an effort to counter dysfunction and disability. Within these models, functional testing represents a standard and validated means of scoring human geriatric function as well as monitoring response to therapy. Because of the great need in dogs, this review aims to provide a reasonable and testable standardized framework for canine functional scoring. We believe a complete assessment of canine geriatric patients should comprise of identifying environmental variables contributing to health status; diagnosing comorbidities related to disease and aging; and characterizing disability with standardized methods. Only through this process can we construct a comprehensive, reasonable, and targeted rehabilitation plan with appropriate follow up aimed at healthy aging.

Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis.

BACKGROUND: The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. RESULTS: We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. CONCLUSION: This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Differential expression of miRNAs and functional role of mir-200a in high and low productivity CHO cells expressing an Fc fusion protein.

OBJECTIVES: We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. RESULTS: Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. CONCLUSION: These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.

Assessment of a Single Intra-Articular Stifle Injection of Pure Platelet Rich Plasma on Symmetry Indices in Dogs with Unilateral or Bilateral Stifle Osteoarthritis from Long-Term Medically Managed Cranial Cruciate Ligament Disease.

PURPOSE: The use of platelet-rich plasma (PRP) to treat canine osteoarthritis has gained support within the scientific community. PRP effects on pain control for degenerative joint disease induced by naturally occurring cranial cruciate ligament instability are limited, particularly in a cohort of dogs with chronic instability and osteoarthritis (>12 months), representing a commonly encountered clinical population that often defaults to medical management. The goal of this study was to assess the effects of a single intra-articular PRP injection into an effected stifle in this cohort, to assess response to treatment, quantitative kinetic data as it relates to percent body weight for peak vertical force (PVF) and vertical impulse (VI) were collected, and symmetry indices related to PVF were determined. METHODS: Twelve dogs with unilateral or bilateral osteoarthritis with ruptured, non-stabilized cranial cruciate ligaments over 12 months duration were identified. Unilateral injections of 2.5 mL of a PRP preparation into the most severely affected stifle based on kinetic analysis was performed. Repeat pressure-sensitive walkway analysis was conducted monthly for 3 months. Peak vertical force (PVF) and vertical impulse (VI) were normalized to body weight and identified in all four limbs. Previously published symmetry indices regarding PVF were calculated, comparing the treated limb with the contralateral limb, ipsilateral forelimb, and contralateral forelimb. RESULTS: After treatment, hind limb symmetry index (SI) regarding PVF showed improved symmetry, suggesting more weight placement at all-time points after injection of the most affected limb (p < 0.01). Further, PVF asymmetry indices assessing contralateral fore (CFL) and hind limb (CHL) as well as ipsilateral forelimb (IFL) revealing a significant decrease from baseline for CHL at week 4 (p = 0.02), but not weeks 8 and 12. The CFL showed decreased differences in symmetry from baseline at each time point (p = 0.03). There were no statistically significant changes in PVF or VI over time in treated dogs. CONCLUSION: A single injection of PRP improved kinetics for minimally 4 weeks with some data suggesting an effect for up to 12 weeks. Therefore, PRP might be a viable therapeutic option for instability and inflammation associated with chronic osteoarthritis due to cranial cruciate ligament disease in the non-surgical patient.

binary junipr: An Interpretable Probabilistic Model for Discrimination.

junipr is an approach to unsupervised learning in particle physics that scaffolds a probabilistic model for jets around their representation as binary trees. Separate junipr models can be learned for different event or jet types, then compared and explored for physical insight. The relative probabilities can also be used for discrimination. In this Letter, we show how the training of the separate models can be refined in the context of classification to optimize discrimination power. We refer to this refined approach as binary junipr. binary junipr achieves state-of-the-art performance for quark-gluon discrimination and top tagging. The trained models can then be analyzed to provide physical insight into how the classification is achieved. As examples, we explore differences between quark and gluon jets and between gluon jets generated with two different simulations.

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq.

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a "high" and "low" clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33-39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.

Successful medical management and rehabilitation of exercise-induced dorsal scapular luxation in an ultramarathon endurance sled dog with magnetic resonance imaging diagnosis of grade II serratus ventralis strain.

Dorsal displacement of the scapula in dogs is rare and often traumatic. This report describes dorsal luxation of the scapula in a sled dog. This case is unique given the injury was sport-related. Magnetic resonance imaging helped direct therapy and monitor healing; and medical management with rehabilitation resulted in full recovery and return to sport. One year after injury, the dog completed both a 482 km and a 1600 km endurance race, placing among the leading teams in the 1600-km race.

Traitement médical réussi d'une luxation scapulaire dorsale aiguë secondaire à la pratique du sport d'endurance chez un chien de traîneau et diagnostic par IRM d'une lésion du m. dentelé ventral. Le déplacement dorsal de la scapula est rare chez le chien et souvent d'origine traumatique. Ce rapport décrit une luxation scapulaire dorsale aiguë chez un chien de traîneau à l'effort. L'intérêt de ce cas tient à sa cause, associé à la pratique de l'activité sportive; à l'analyse des changements en IRM soutenant une thérapie ciblée consistant en un support médical ainsi que d'un programme de rééducation physique, résultant en un rétablissement complet. Un an après cette blessure, le chien termina des courses d'endurance de 482 km et de 1600 km, se classant lors de cette dernière parmi les meilleures équipes de la course.(Traduit par les auteurs).

Pharmacokinetics, Safety, and Clinical Efficacy of Cannabidiol Treatment in Osteoarthritic Dogs.

Objectives: The objectives of this study were to determine basic oral pharmacokinetics, and assess safety and analgesic efficacy of a cannabidiol (CBD) based oil in dogs with osteoarthritis (OA). Methods: Single-dose pharmacokinetics was performed using two different doses of CBD enriched (2 and 8 mg/kg) oil. Thereafter, a randomized placebo-controlled, veterinarian, and owner blinded, cross-over study was conducted. Dogs received each of two treatments: CBD oil (2 mg/kg) or placebo oil every 12 h. Each treatment lasted for 4 weeks with a 2-week washout period. Baseline veterinary assessment and owner questionnaires were completed before initiating each treatment and at weeks 2 and 4. Hematology, serum chemistry and physical examinations were performed at each visit. A mixed model analysis, analyzing the change from enrollment baseline for all other time points was utilized for all variables of interest, with a p ≤ 0.05 defined as significant. Results: Pharmacokinetics revealed an elimination half-life of 4.2 h at both doses and no observable side effects. Clinically, canine brief pain inventory and Hudson activity scores showed a significant decrease in pain and increase in activity (p < 0.01) with CBD oil. Veterinary assessment showed decreased pain during CBD treatment (p < 0.02). No side effects were reported by owners, however, serum chemistry showed an increase in alkaline phosphatase during CBD treatment (p < 0.01). Clinical significance: This pharmacokinetic and clinical study suggests that 2 mg/kg of CBD twice daily can help increase comfort and activity in dogs with OA.

Serum myoglobin, creatine kinase, and cell-free DNA in endurance sled dogs and sled dogs with clinical rhabdomyolysis.

OBJECTIVE: To evaluate whether cell-free DNA (cfDNA) concentrations are increased in dogs with exertional rhabdomyolysis and whether concentrations are correlated with serum myoglobin concentration and creatine kinase activity. DESIGN: Observational cohort study. SETTING: Yukon Quest 1,000-mile International Sled Dog Race 2015. ANIMALS: Twelve normal competitive sled dogs; 5 dogs with rhabdomyolysis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was collected from all confirmed cases of exertional rhabdomyolysis and compared to the winning team at the midrace point. Results indicate that median cfDNA did not increase, but decreased by the race finish (prerace = 314.2 ng/mL versus midrace = 283.7 ng/mL versus postrace = 249.5 ng/mL). There were no rises in median cfDNA in dogs with rhabdomyolysis (255 ng/mL) negating its potential utility as a measure of acute skeletal muscle compromise. In contrast, myoglobin concentration and creatine kinase activity at the midrace point for normal dogs were significantly lower than dogs with rhabdomyolysis. Values for myoglobin and creatine kinase were strongly positively correlated (R = 0.91). CONCLUSIONS: cfDNA is not a useful biomarker for exertional rhabdomyolysis in contrast to myoglobin and creatine kinase. Further evaluation of timing and clinical signs suggests that exertional rhabdomyolysis occurs early in endurance activities. Among the dogs with rhabdomyolysis, the dog that demonstrated clinical signs had the highest serum creatine kinase activity and myoglobin concentration.

Clonal variation in productivity and proteolytic clipping of an Fc-fusion protein in CHO cells: Proteomic analysis suggests a role for defective protein folding and the UPR.

Product degradation, such as clipping, is a common quality issue in the production of Fc-fusion proteins from Chinese hamster ovary (CHO) cells. Degradation of proteins is mainly due to the action of either intracellular or extracellular host cell proteases. This study was carried out to understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of Fc-fusion protein clipping. The cell lines that displayed the highest levels of clipping also produced less product than the cell lines with a lower level of clipping. In this study we applied differential quantitative label-free LC-MS/MS proteomic analysis to group clonally-derived cell lines (CDCLs) based on the level of clipping of the Fc-fusion protein. The analysis was carried out over two times points in culture and clones were designated as either having 'high' or 'low' clipping phenotypes. We have identified 200 differentially expressed proteins using quantitative label-free LC-MS/MS analysis between the two experimental groups. Functional assessment of the resultant proteomic data using Gene Ontology analysis showed a significant enrichment of biological processes and molecular functions related to protein folding, response to unfolded protein and protein translation. The levels of several proteases were also increased. This study identified protein targets that could be modified using cell line engineering approaches to improve the quality of recombinant Fc-fusion protein production in the biopharmaceutical industry.

Effective and efficient characterization of Chinese hamster ovary production cell lines using automated intracellular staining and statistical modeling.

Mammalian cell line development is critical to bioproduct manufacturing. Success requires selecting a line with desirable performance characteristics, including consistent expression throughout the proposed manufacturing window. Given the genetic and phenotypic flux inherent to immortalized lines such as Chinese hamster ovary cells, clonally-derived cell line characterization is vital. We describe here the development and implementation of a novel addition to our characterization approach to ensure production cell line suitability: automated intracellular staining with statistical modeling. Case studies are presented which highlight this method's sensitivity to epigenetic expression effects, closing a gap left by our historically-leveraged genetic suitability characterization. Additionally, we demonstrate how an orthogonal, complimentary assay can help identify opportunities for improvement in even a well-established methodology such as our genetic suitability assessment. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:570-583, 2018.

Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses.

Oxidation of monoclonal antibodies (mAb) is a common chemical modification with potential impact on a therapeutic protein's activity and immunogenicity. In a previous study, it was found that tryptophan oxidation (Trp-ox) levels of two mAb produced in Chinese hamster ovary (CHO) cells were significantly lowered by modifying cell culture medium/feed. In this study, transcriptome analysis by RNA-Seq is applied to further elucidate the underlying mechanism of those changes in lowering the Trp-ox levels. Cell samples from the 5L fed-batch conditions are harvested and subjected to RNA-Seq analysis. The results showed that the cell culture changes had little impact on neither the expression of the mAb transgenes nor genes related to glycosylation. However, those changes did significantly alter the expression of multiple genes (p-value ≤0.05 and absolute fold change ≥1.5 or adjusted p-value ≤0.1) involved in transport of copper, regulation of glutathione, iron storage, heme reduction, oxidative phosphorylation, and Nrf2-mediated antioxidative response. These findings suggest a key underlying mechanism in lowering Trp-ox levels by CDM was likely to be collectively controlling ROS levels through regulation of those genes' expression. This is the first example, to our knowledge, applying transcriptomic analysis to mechanistically understand the impact of cell culture on mAb oxidation.

Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.