Background: Aggregated biotherapeutics have the potential to induce an immune response.Results: Aggregates can enhance innate and adaptive immune responses of PBMC.Conclusion: The response depends on aggregate type, immunogenicity of the monomer, donor immune status, and high particle numbers in the in vitro assay.Significance: This is the first study showing the impact of aggregate characteristics on the potential immune response of PBMC.
An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.
An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size enriched into different size bins by low speed centrifugation or a combination of gravitational sedimentation and Fluorescence-Activated Cell Sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5–10 μm in size displayed elevated cytokine release profiles compared to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by Microflow imaging, Transmission Electron Microscopy, and Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in-vitro PBMC studies to rank order the immunogenic potential of various types of mAb particles is discussed.
A potential risk factor for immunogenicity of a biotherapeutic is the low levels of host cell protein (HCP) impurities that remain in the product following the purification process. During process development, significant attention has been devoted to removing HCPs due to their potential safety risk. Samples from different purification steps of several monoclonal antibodies (mAbs) purified by one type of platform were evaluated for their residual Chinese Hamster Ovary (CHO) cell-derived HCP content. HCPs in both in-process (high levels of HCP) and highly purified (low levels of HCP) samples were identified and quantitated by proteomic analysis via mass spectrometry. The responses to HCPs were evaluated in an in vitro assay using PBMC from a population of healthy and disease state individuals. Results indicated that samples with up to 4000 ppm HCP content (levels 200 times greater than the drug substance) did not pose a higher immunogenicity risk than highly purified mAb samples. As an orthogonal method to predict immunogenicity risk, in silico algorithms that probe amino acid sequence for foreign epitope content were used to evaluate over 20 common HCPs (identified in the different mAb samples). Only a few HCPs were identified as high risk by the algorithms; however, the in vitro assay results indicated that the concentration of these HCPs from in-process biotherapeutic mAb samples was not sufficient to stimulate an immune response. This suggests that high levels of HCP in mAb biotherapeutics purified by this type of platform do not increase the potential risk of immunogenicity of these molecules. Insights from these studies can be applied to HCP control and risk assessment strategies.
BackgroundAmong the 1.21 billion population of India, 26.8 million individuals have disabilities, and around five million are visually impaired. These children encounter problems related to oral health maintenance, thus further leading to dental problems. Even though Pediatric dentists treat visually impaired children for their oral problems, they emphasize mainly on the prevention of dental caries. Dental caries has a multifactorial etiology, and dentists are unable to find a complete solution for its prevention. However, reducing Streptococcus mutans has been seen to reduce the caries rate in the past. The use of the herbal product Emblica officinalis to reduce Streptococcus mutans levels has been documented. An affordable delivery system is required to use Emblica officinalis for its anticaries action. Thus, a unique delivery system of herbal sugar-free lollipops containing Emblica officinalis extract was made and can effectively deliver antimicrobial action in visually impaired children. AimTo evaluate the antibacterial efficacy of Emblica officinalis lollipop on Streptococcus mutans counts and pH levels in institutionalized visually impaired children. MethodA total of 60 institutionalized visually impaired children (age: 4 to 14 years) were selected. The study consisted of two groups (experimental "Emblica officinalis lollipop" and control "placebo lollipop"), and the children were divided equally into both groups. Children from the study and control groups were subjected to the respective lollipops twice daily for seven days. Streptococcus mutans count and pH count were evaluated at baseline and after seven days post-intervention of the respective lollipop. ResultsThe results showed that in both groups, Streptococcus mutans count was reduced post-intervention. However, the efficacy of the study group (Emblica officinalis lollipop) in inhibiting the Streptococcus mutans count was better than the control group (placebo lollipop). An increase in the pH level was seen postintervention for both the study and control groups. And on the intergroup comparison, no statistical significance was found. ConclusionThe use of Emblica officinalis lollipop is effective in inhibiting the Streptococcus mutans count when compared with the placebo lollipop. While marginal pH change was seen in both groups. Thus, the herbal modality most acceptable without any pharmaceutical concerns should be chosen. Emblica officinalis lollipops can be used in institutionalized visually impaired children to reduce the oral Streptococcus mutans count and maintain a healthy oral cavity.
Background Dental anxiety has been a major concern for dentists while operating children. For a smooth, uneventful treatment, operators must incorporate various behavior management techniques in their practice. The incorporation of magic tricks as behavior management techniques has been used earlier by physicians and nurses to reduce pre-operative anxiety in hospitals. This study aimed to compare the impacts of magic tricks on reducing dental anxiety in children. Material and methods Patients aged four to 11 years were eligible for inclusion. The study comprised two groups of 15 children each. During the first visit, children weren’t subjected to any behavior management. Behavior management aids (magic tricks and audiovisuals) were used during the second visit. Hemodynamic parameters along with an anxiety scale were used to assess anxiety in children before, during, and after treatment procedures. Venham’s picture test and modified visual analog scale were also used to assess the anxiety. Results A reduction in anxiety was seen in both groups after behavior management was used. The hemodynamic parameters like blood pressure and pulse rate were seen to decrease during the second visit, while the oxygen saturation was seen to increase. Conclusion The study demonstrates that magic trick along with audiovisual aids was effective in controlling dental anxiety. Thus, magic tricks can be used in dental practice as a behavior management aid for children to facilitate cooperative behavior. Reducing a child's dental anxiety through various different magic trick aids could be a potential behavior management modality that needs further research.
To evaluate antibacterial efficacy of Triphala toothwipes on oral Streptococcus mutans counts in intellectually disabled (ID) children. Methods: Twenty-seven children with a mild ID were randomly divided into two groups: A -Triphala and B -placebo group. Toothwipes were given to caregivers of children belonging to the respective groups, and were instructed to use them 1 h after their meals, twice a day for 7 days. Simplified Plaque index was recorded and plaque samples were collected for microbiological examination at baseline, 48 h, and 7 days. The obtained data were tabulated and analyzed.Results: Triphala group showed a statistically significant reduction of S. mutans after 48 h and 7 days. Both Triphala and placebo groups showed a statistically significant reduction in dental plaque after 48 h and 7 days. Conclusion:Triphala toothwipes are effective against the oral S. mutans compared with placebo toothwipes, while both the toothwipes are equally effective in reducing dental plaque. Thus, Triphala toothwipes can be used as an adjunct aid along with routine oral hygiene practices in individuals who lack psychomotor skills or are dependent on others to maintain oral hygiene, including infants, preschoolers, geriatric population, and children with special health care needs.
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