Evaluation of the In-Use Stability of Monoclonal Antibody IV Admixtures Prepared from Drug Products Containing Polysorbate 20 Degraded by Host-Cell Lipases.

Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.

Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

Prediction of long-term polysorbate degradation according to short-term degradation kinetics.

Polysorbates (PSs) are a class of surfactants commonly used in the formulation of protein therapeutic agents to provide protection against denaturation and aggregation. When the PS in these drug formulations degrades, loss of stabilization of the protein therapeutic and formulation may occur, resulting in particulate formation or other undesirable changes in product critical quality attributes. Here, we present a simplified platform to predict long-term PS20 and PS80 degradation for monoclonal antibody drugs containing the PS-degrading enzyme lysosomal acid lipase. The platform was based on a temperature-dependent equation derived from existing PS20 degradation stability data. Accurate prediction of both PS20 and PS80 hydrolysis for as long as 2 years was achieved through short-term kinetics studies performed within 2 weeks. This platform substantially shortens the time required to determine the long-term stability of PS degradation and therefore can be used to guide the purification process and optimization of antibody formulations.

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody.

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pKa on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.

Container Closure and Delivery Considerations for Intravitreal Drug Administration.

Intravitreal (IVT) administration of therapeutics is the standard of care for treatment of back-of-eye disorders. Although a common procedure performed by retinal specialists, IVT administration is associated with unique challenges related to drug product, device and the procedure, which may result in adverse events. Container closure configuration plays a crucial role in maintaining product stability, safety, and efficacy for the intended shelf-life. Careful design of primary container configuration is also important to accurately deliver small volumes (10-100 µL). Over- or under-dosing may lead to undesired adverse events or lack of efficacy resulting in unpredictable and variable clinical responses. IVT drug products have been traditionally presented in glass vials. However, pre-filled syringes offer a more convenient administration option by reducing the number of steps required for dose preparation there by potentially reducing the time demand on the healthcare providers. In addition to primary container selection, product development studies should focus on, among other things, primary container component characterization, material compatibility with the formulation, formulation stability, fill volume determination, extractables/leachables, and terminal sterilization. Ancillary components such as disposable syringes and needles must be carefully selected, and a detailed administration procedure that includes dosing instructions is required to ensure successful administration of the product. Despite significant efforts in improving the drug product and administration procedures, ocular safety concerns such as endophthalmitis, increased intraocular pressure, and presence of silicone floaters have been reported. A systematic review of available literature on container closure and devices for IVT administration can help guide successful product development.

Influence of exercise modality on cardiac parasympathetic and sympathetic indices during post-exercise recovery.

OBJECTIVES: This study investigated indirect measures of post-exercise parasympathetic reactivation (using heart-rate-variability, HRV) and sympathetic withdrawal (using systolic-time-intervals, STI) following upper- and lower-body exercise. DESIGN: Randomized, counter-balanced, crossover. METHODS: 13 males (age 26.4±4.7years) performed maximal arm-cranking (MAX-ARM) and leg-cycling (MAX-LEG). Subsequently, participants undertook separate 8-min bouts of submaximal HR-matched exercise of each mode (ARM and LEG). HRV (including natural-logarithm of root-mean-square-of-successive-differences, Ln-RMSSD) and STI (including pre-ejection-period, PEP) were assessed throughout 10-min seated recovery. RESULTS: Peak-HR was higher (p=0.001) during MAX-LEG (182±7beatsmin-1) compared with MAX-ARM (171±12beatsmin-1), while HR (p<0.001) and Ln-RMSSD (p=0.010) recovered more rapidly following MAX-ARM. PEP recovery was similar between maximal bouts (p=0.106). HR during submaximal exercise was 146±7 (LEG) and 144±8beatsmin-1 (LEG) (p=0.139). Recovery of HR and Ln-RMSSD was also similar between submaximal modalities, remaining below baseline throughout recovery (p<0.001). PEP was similar during submaximal exercise (LEG 70±6ms; ARM 72±9ms; p=0.471) although recovery was slower following ARM (p=0.021), with differences apparent from 1- to 10-min recovery (p≤0.036). By 10-min post-exercise, PEP recovered to baseline (132±21ms) following LEG (130±21ms; p=0.143), but not ARM (121±17ms; p=0.001). CONCLUSIONS: Compared with submaximal lower-body exercise, HR-matched upper-body exercise elicited a similar recovery of HR and HRV indices of parasympathetic reactivation, but delayed recovery of PEP (reflecting sympathetic withdrawal). Exercise modality appears to influence post-exercise parasympathetic reactivation and sympathetic withdrawal in an intensity-dependent manner. These results highlight the need for test standardization and may be relevant to multi-discipline athletes and in clinical applications with varying modes of exercise testing.

Longer exercise duration delays post-exercise recovery of cardiac parasympathetic but not sympathetic indices.

PURPOSE: This study investigated non-invasive indices of post-exercise parasympathetic reactivation (using heart rate variability, HRV) and sympathetic withdrawal (using systolic time intervals, STI) following different exercise durations. METHODS: 13 healthy males (age 26.4 ± 4.7 years) cycled at 70% heart rate (HR) reserve for two durations-8 min (SHORT) and 32 min (LONG)-on separate occasions: HRV (including natural logarithm of root mean square of successive differences, Ln-RMSSD) and STI (including pre-ejection period, PEP) were assessed throughout 10 min seated recovery. RESULTS: Exercise HR was similar between SHORT and LONG (146 ± 7 and 147 ± 6 b min-1, respectively; p = 0.173), as was HR deceleration during 10 min recovery (p = 0.199). HR remained elevated above baseline (p < 0.001) throughout recovery for both trials (SHORT 82 ± 13 b min-1; LONG 86 ± 10 b min-1, at 10 min post-exercise). Ln-RMSSD was similar at end-exercise between trials (SHORT 1.10 ± 0.30 ms; LONG 1.05 ± 0.73 ms; p = 0.656), though it recovered more rapidly following SHORT (p = 0.010), with differences apparent from 1 min (SHORT 2.29 ± 1.08 ms; LONG 1.85 ± 0.82 ms; p = 0.005) to 10 min post-exercise (SHORT 2.89 ± 0.80 ms; LONG 2.46 ± 0.70 ms; p = 0.007). Ln-RMSSD remained suppressed below baseline throughout recovery following both trials (p < 0.001). PEP was the same at end exercise for both trials (70 ± 6 ms), with exercise duration having no effect on recovery (p = 0.659). By 10 min post-exercise, PEP increased to 130 ± 21 ms (SHORT) and 131 ± 20 ms (LONG), which was similar to baseline (p ≥ 0.143). CONCLUSIONS: Prolonged exercise duration attenuated the recovery of HRV indices of parasympathetic reactivation, but did not influence STI indices of sympathetic withdrawal. Therefore, duration must be considered when investigating post-exercise HRV. Monitoring these measures simultaneously can provide insights not revealed by underlying HR or either measure alone.

Cardiac Autonomic Responses during Exercise and Post-exercise Recovery Using Heart Rate Variability and Systolic Time Intervals-A Review.

Cardiac parasympathetic activity may be non-invasively investigated using heart rate variability (HRV), although HRV is not widely accepted to reflect sympathetic activity. Instead, cardiac sympathetic activity may be investigated using systolic time intervals (STI), such as the pre-ejection period. Although these autonomic indices are typically measured during rest, the "reactivity hypothesis" suggests that investigating responses to a stressor (e.g., exercise) may be a valuable monitoring approach in clinical and high-performance settings. However, when interpreting these indices it is important to consider how the exercise dose itself (i.e., intensity, duration, and modality) may influence the response. Therefore, the purpose of this investigation was to review the literature regarding how the exercise dosage influences these autonomic indices during exercise and acute post-exercise recovery. There are substantial methodological variations throughout the literature regarding HRV responses to exercise, in terms of exercise protocols and HRV analysis techniques. Exercise intensity is the primary factor influencing HRV, with a greater intensity eliciting a lower HRV during exercise up to moderate-high intensity, with minimal change observed as intensity is increased further. Post-exercise, a greater preceding intensity is associated with a slower HRV recovery, although the dose-response remains unclear. A longer exercise duration has been reported to elicit a lower HRV only during low-moderate intensity and when accompanied by cardiovascular drift, while a small number of studies have reported conflicting results regarding whether a longer duration delays HRV recovery. "Modality" has been defined multiple ways, with limited evidence suggesting exercise of a greater muscle mass and/or energy expenditure may delay HRV recovery. STI responses during exercise and recovery have seldom been reported, although limited data suggests that intensity is a key determining factor. Concurrent monitoring of HRV and STI may be a valuable non-invasive approach to investigate autonomic stress reactivity; however, this integrative approach has not yet been applied with regards to exercise stressors.

Higher exercise intensity delays postexercise recovery of impedance-derived cardiac sympathetic activity.

Systolic time intervals (STIs) provide noninvasive insights into cardiac sympathetic neural activity (cSNA). As the effect of exercise intensity on postexercise STI recovery is unclear, this study investigated the STI recovery profile after different exercise intensities. Eleven healthy males cycled for 8 min at 3 separate intensities: LOW (40%-45%), MOD (75%-80%), and HIGH (90%-95%) of heart-rate (HR) reserve. Bio-impedance cardiography was used to assess STIs - primarily pre-ejection period (PEP; inversely correlated with cSNA), as well as left ventricular ejection time (LVET) and PEP:LVET - during 10 min seated recovery immediately postexercise. Heart-rate variability (HRV), i.e., natural-logarithm of root mean square of successive differences (Ln-RMSSD), was calculated as an index of cardiac parasympathetic neural activity (cPNA). Higher preceding exercise intensity elicited a slower recovery of HR and Ln-RMSSD (p < 0.001), and these measures did not return to baseline by 10 min following any intensity (p ≤ 0.009). Recovery of STIs was also slower following higher intensity exercise (p ≤ 0.002). By 30 s postexercise, higher preceding intensity resulted in a lower PEP (98 ± 14 ms, 75 ± 6 ms, 66 ± 5 ms for LOW, MOD, and HIGH, respectively, p < 0.001). PEP recovered to baseline (143 ± 11 ms) by 5 min following LOW (139 ± 13 ms, p = 0.590) and by 10 min following MOD (145 ± 17 ms, p = 0.602), but was still suppressed at 10 min following HIGH (123 ± 21 ms, p = 0.012). Higher preceding exercise intensity attenuated the recovery of indices for cSNA (from STIs) and cPNA (from HRV) in a graded dose-response fashion. While exercise intensity must be considered, acute recovery may be a valuable period during which to concurrently monitor these noninvasive indices, to identify potentially abnormal cardiac autonomic responses.

Depression, Anxiety, and Alcohol Use in Elite Rugby League Players Over a Competitive Season.

OBJECTIVE: To assess the prevalence of symptoms of depression, anxiety, and rates of alcohol misuse in elite rugby league players in Australasia. DESIGN: A cross-sectional, epidemiological study with repeated measures. SETTING: Surveys were conducted during the 2015 preseason and in-season. PARTICIPANTS: Four hundred four elite rugby league players participated preseason and 278 players in-season. MAIN OUTCOME MEASURES: Symptoms of depression were measured using the Patient Health Questionnaire-9 scale, symptoms of generalized anxiety disorder (GAD) with the GAD-7 scale, and the Alcohol Use Disorders Identification Test Consumption scale was used to assess hazardous alcohol use. RESULTS: The overall prevalence of depression was 12.6% preseason and 10.1% in-season. Generalized anxiety disorder had a prevalence of 14.6% and 10.1% for these 2 periods. Overall, 68.6% of players had hazardous levels of alcohol use preseason, and 62.8% in-season. There was no significant difference for any of the main outcomes between the periods. Players with a history of mental illnesses had 5.62 greater odds (95% confidence interval [CI], 2.62-12.04) of depression than those without during preseason, and 22.08 greater odds (95% CI, 7.77-62.71) in-season. Players reporting ≥3 previous concussions had 2.02 greater odds (95% CI, 1.07-3.82) of depression than those reporting ≤2 in the preseason sample. CONCLUSIONS: Rugby league players have a lower prevalence of depression compared with studies of the general population and other athletes, but a higher prevalence of GAD, and high rates of alcohol misuse. Clubs may consider implementing regular screening for these conditions. Further prospective research to determine causality of independent factors is required.

Low-intensity pulsed ultrasound in lower limb bone stress injuries: a randomized controlled trial.

OBJECTIVE: To evaluate the effectiveness of low-intensity pulsed ultrasound (LIPUS) for the improvement of lower limb bone stress injuries in a civilian population. DESIGN: A prospective, randomized, double-blinded, placebo-controlled trial to compare LIPUS with placebo. SETTING: Civilian private practice population in Sydney, Australia. PARTICIPANTS: Subjects were recruited if a grade II-IV bone stress injury was diagnosed on magnetic resonance imaging (MRI) of either the postero-medial tibia, fibula or second, third, or fourth metatarsal. Subjects of all levels of sporting activity were included. Thirty subjects were initially recruited, and 23 subjects were included in the final analysis. INTERVENTIONS: Subjects were randomized into either the treatment or placebo arm and matched to the site of injury (tibia, fibula, or metatarsal). Subjects in both arms used either treatment or placebo devices for 20 minutes daily for 4 weeks. MAIN OUTCOME MEASURES: Six clinical parameters (night pain, pain at rest, pain on walking, pain with running, tenderness, and pain with single leg hop) were compared before and after intervention. The changes in MRI grade and bone marrow edema size were also compared. RESULTS: There were no significant differences between the treatment and placebo conditions for changes in MRI grading (2.2 vs 2.4, P = 0.776) or bone marrow edema size (3 vs 4.1, P = 0.271). There were no significant differences between the treatment and placebo conditions for the 6 clinical parameters. CONCLUSIONS: Low-intensity pulsed ultrasound was found not to be an effective treatment for the healing of lower limb bone stress injuries in this study. However, this was measured over a relatively short duration of 4 weeks in a small, mostly female population. CLINICAL RELEVANCE: This double-blinded, randomized, placebo-controlled trial has shown that LIPUS is not an effective treatment for lower limb bone stress injuries.

What is the effect of compression garments on a balance task in female athletes?

OBJECTIVES: To investigate the effect of long leg compression garments on the postural sway and balance ability of female athletes at a state sports institute. DESIGN: A laboratory was set up to analyse kinetic and kinematic variables using a double blind, randomised controlled repeated measures cross over design. METHOD: Participants were required to perform a single leg balance task for up to 60s across six conditions; including eyes-open and eyes-closed while wearing conventional shorts (control), loose-fitted compression garment and well-fitted compression garments. Simultaneous measurements of ground reaction forces and full body joint kinematics were recorded. Postural stability was assessed by measuring the overall stabilisation time as well as the movement of the centre of pressure (CoP) and centre of mass (CoM) from baseline measures. RESULTS: During one leg stance, significantly greater postural stability (p < 0.01) was observed with eyes open vs eyes closed, irrespective of compression group. A significantly greater (p < 0.05) balance time was observed with eyes closed when wearing well-fitted compression garments compared to conventional shorts. Differences were not present with use of the loose-fitted garment. Additionally, a significant interaction effect between compression condition and vision was observed analysing the variation about the sway (swaySD) of the CoP and CoM data (p < 0.05). The interaction effect revealed greater variability of movement with eyes closed as participants' level of compression decreased. No significant differences were observed with eyes open. CONCLUSIONS: The difficulties of postural stability while maintaining the single leg stance wearing conventional shorts were improved with use of the well-fitted compression garments (in the eyes-closed condition). Proper fitted compression garments may be beneficial for injury management and injury prevention.

The effect of prior endurance training on nap sleep patterns.

PURPOSE: The use of daytime napping as a recovery tool following exercise is virtually unexplored. The objective of this study was to assess the quality of daytime nap sleep following endurance training in an athletic population, and to appraise the optimal circadian timing of the nap and the time interval between training and the nap. METHODS: Six physically trained male subjects (22.5+/-2.4 y) performed four separate standardized 90-min endurance training sessions followed by a 90-min daytime nap either 1 or 2 h after training (time interval), commencing at either 10:30 or 11:30 (circadian timing). During the nap, sleep was monitored using polysomnography. Subjective measurements of sleep quality, alertness and preparedness to train following a nap were recorded using a visual analog scale. RESULTS: The duration of slow wave sleep (SWS) was significantly greater during the 11:30 naps (13.7+/-9.0 min) compared with the 10:30 naps (6.9+/-8.8 min) (P=.049). There was no significant difference in SWS duration between a 1-h (10.6+/-10.2 min) or 2-h (10.0+/-9.0 min) time interval between training and the nap (P=.82). No other sleep variables differed significantly according to circadian timing or time interval. CONCLUSION: Recovery naps commenced later in the morning contain more SWS than earlier naps. The data imply that daytime naps have a potential role as a valuable recovery tool following endurance exercise, given the suggested energy restorative functions of SWS.