Search

Design of experiments (DoE) is used in process development to optimise the operating conditions of unit operations in a cost-effective and time-saving manner. Along with high throughput technologies, the modern high throughput process development lab can turnover a tremendous amount of data with minimal feedstock. These benefits are most useful when applied to the purification bottleneck, which accounts for up to 80% of the total process operating costs. However due to complexities of biochemical reactions and the large number interacting factors in unit operations (which usually cross interact with each other), even carefully planned DoE experiments on high throughput platforms can become difficult to manage and/or not provide useful information. This thesis examines the simplex search method and develops a set of protocols for use of the search method in combination with traditional DoE experimental design protocols. It is that is demonstrated in the developed in chapter 3 whilst also optimising a ammonium sulphate based precipitation step of an industrially relevant feedstock. Comparisons were drawn between a high resolution brute force study, a response surface DoE, the simplex method and then a combination of DoE and the simplex method. Various strategies were demonstrated that get the most out of the simplex method and mitigate against potential pitfalls. The precipitation step was optimised for yield and purity over the 3 factors, pH, ammonium sulphate concentration and initial MAb concentration and the results showed the simplex method was capable of rapidly identifying the optimum conditions in a very large 3 factor design space on an average of 18 experiments. The expansive study not only served as a testing ground for the methods comparison but demonstrated precipitation as a high throughput, low cost substitute for the expensive Protein A step. The DoE –simplex search protocols are then refined in two complex case studies in chapter 4, a PEG precipitation primary capture step and an ammonium sulphate precipitation and centrifugation sequence. The five factor precipitation and centrifugation sequence was especially complicated and utilised ultrascale down models provide accurate scale up data. This involved calibrating an acoustic device to provide shear treatment to the precipitate pre-centrifugation and using jet mixing equations to correlate precipitate conditioning between the TTecan robot’s tips and an impeller in a stirred tank. The techniques developed were all applicable to microscale and high throughput. In both instances, the combined DoE-simplex approach retuned superior results both in terms of experimental savings and generating information-rich data from the final local regions DoE around the simplex located optimums. A microscale chromatography protocol was developed on the Tecan liquid handling robot and demonstrated on screening work with different Protein A and cation exchange media. The caveats encountered when creating the running methods and the analytical methods supporting it for the Atoll robocolumns were highlighted and mitigation solutions implemented. The automated microscale Protein A method was successfully scaled up 50x from a 200 μL robocolumn to a conventional 10 mL labscale column. After selecting a cation exchange resin for developing an aggregate removal step, the DoE-simplex methodology was applied to an antibody product with an extremely high aggregate level and a comparison optimisation was made with a central composite design DoE. The difficult four factor design space overwhelmed the DoE and having used more experiment numbers than the DoE-simplex methodology, only went as far to show the high levels of curvature in the system and offer a poor prediction of the surface. The DoE-simplex methodology was able to provide a general model of the whole surface from the DoE, locate the optimum with the simplex in fewer experiment numbers. This subsequently allowed a local DoE to be applied to the optimum region to determine a robust operating range for the cation exchange step.

Treadmill running has historically been utilized in the laboratory to mimic outdoor running. Recent developments in portable technology, such as Inertial Measurement Units (IMUs) allow researchers to assess runners in their natural environment. PURPOSE: The primary purpose of the study was to compare peak tibial acceleration, stance time, stride frequency, and rearfoot eversion velocity between treadmill and overground running (asphalt, track, and grass). The secondary purpose was to test the reliability of IMU-based estimations of maximum rearfoot eversion velocity during treadmill running. METHODS: Twenty subjects (Age: 22.1 ± 2.0 yrs, Mass: 70.8 ± 11.9 kg, Height: 174.5 ± 10.0 cm, 8F/12M) participated. After consent and a warm-up period, IMUs were placed on the anteromedial aspect of the right distal tibia (1600Hz) and the posterior heel cap of the right shoe (1125 Hz) to record 3D linear accelerations and angular velocities. Subjects then ran three 30-meter trials on each overground surface (grass, track, and asphalt) at their self-selected speed. A timing system was used to ensure the same running speed was used between conditions. Subsequently, all subjects ran on a treadmill while their rearfoot motion was recorded through high-speed videography (240 Hz). Only one continuous trial was performed for each subject on the treadmill. In each of these trials, the variables of stride frequency, tibial acceleration, maximum rearfoot eversion velocity, and stance time were collected from the IMUs. Maximum rearfoot eversion velocity was the only variable processed from the rearfoot motion video data. A total of 30 steps from each condition were extracted and analyzed (10 steps from each trial for overground surfaces). In our statistical analysis, separate repeated measures ANOVAs or Friedman tests were performed on the mean and variability of each variable, depending on data normality, to examine differences between surface conditions. Post-hoc analysis was performed when appropriate through either Fisher’s LSD (α = 0.05) or Wilcoxon signed-rank tests (α = 0.008). RESULTS: The means of stride frequency, peak tibial acceleration, and maximum rearfoot eversion velocity were significantly different (p < 0.001) between surfaces. Specifically, stride frequency was the fastest during treadmill running (1.39 (0.09) strides/sec) and slowest while running on grass (1.35 (0.07) strides/sec). Peak tibial acceleration was not different between the outdoor running conditions (asphalt: 11.0 (2.7) G, grass: 10.7 (3.4) G, and track: 10.6 (2.3) G), but significantly less during treadmill running (7.9 (1.6) G). Maximum rearfoot eversion velocity was lowest on grass (394.9 (256.5) deg/s) and greatest on track (623.5 (299.3) deg/s) and asphalt (620.7 (289.1) deg/sec). There was no difference in stance time between surfaces (p = 0.231). The variabilities of stance time, stride frequency, maximum rearfoot eversion velocity, and peak tibial acceleration were all found to be significantly different (p < 0.001) between running surface. Treadmill running presented with the lowest levels of variability in stride frequency (0.016 (0.005) strides/sec), maximum rearfoot eversion velocity (70.10 (35.58) deg/s), and peak tibial acceleration (0.79 (0.32) G) across all conditions. Due to this, all variables other than stance time were significantly less variable during treadmill running than any of the overground conditions. In contrast, running on grass displayed significantly larger variabilities across all conditions in stance time (0.014 (0.009) sec), stride frequency (0.029 (0.010) strides/sec), maximum rearfoot eversion velocity (123.71 (36.16) deg/s), and peak tibial acceleration (2.41 (1.21) G). Lastly, maximum rearfoot eversion velocities acquired from the heel mounted IMU obtained a moderate reliability with the high-speed videography (ICC = 0.739, CI: 0.322 – 0.899). CONCLUSION: A single IMU appears to only be moderately reliable when recording eversion velocities during running, so caution may be warranted when using a single IMU to estimate rearfoot motion during running. With the use of IMUs, treadmill running has been shown to be different than overground in terms of both mean differences and variability. As a result, laboratory-based studies on running biomechanics may not truly reflect the “natural” gait used on outdoor running surfaces.

PURPOSE: The purpose of this study was to examine the use of the ActiGraph GT9X gyroscope and magnetometer for turn detection and quantifying turn degree during walking and running. METHODS: Participants (N=17) completed pivot trials, treadmill walking and running (3 to 6 mph) and different degrees of turns (i.e. 45°, 90°, 135°, and 180°) during over-ground walking and running. Pivot and walking and running activities were completed for 1-minute and 6-minutes per trial, respectively. Turn frequency was constant (10 turns/minute) across all overground walking and running trials. ActiGraph GT9X devices were placed on the left and right hips, wrists, and ankles and a Cosmed K4b2 measured energy expenditure. Raw ActiGraph GT9X gyroscope and magnetometer data were processed through various low-pass filter frequencies (0.25 Hz to 2.0 Hz). Treadmill and pivot trials were used to develop thresholds for turn detection using gyroscope and magnetometer data. Cross-validation was completed using over-ground trials for turn detection and turn degree using filtered gyroscope data. Cosmed data (VO2) were averaged over 30-second periods and then converted to relative VO2 (ml/kg.min) for each activity. Linear mixed models were used to compare actual and predicted number of turns, measured and predicted turn degree, and differences in VO2 across walking and running speed and turn degree. Linear regression models were used to predict VO2 using speed, turn degree, and speed and turn degree. RESULTS: Greater than 98% of turns were detected when using gyroscope data filtered at 0.25 Hz. Turn degree was estimated within 2.2° of measured turn degree across all speeds. In general, the VO2 of walking and running increased as the turn degree increased beyond 135°. Walking and running speed explained 83.8% of the variability in VO2, and the addition of turn degree explained an additional 4.3%. CONCLUSION: The ActiGraph GT9X gyroscope, when filtered at 0.25 Hz can be used to detect the number of turns and estimate turn degree. The magnetometer was only useful for detecting the number of turns. Future work is needed to explore the gyroscope application for turn detection performance during activities other than walking and running and estimating VO2.

INTRODUCTION: Tightness of the hip flexor muscle group may contribute to altered sagittal plane kinematics of the lumbo-pelvic-hip (LPH) complex during dynamic movements. Therefore, the purpose of this study is to analyze the effects of a three-week home-based stretching program on passive hip extension (PHE), as well as on active hip extension (AHE), anterior pelvic tilt (APT), and lumbar spine extension (LSE) when running. METHODS: Twenty healthy subjects with limited PHE underwent a 3D gait analysis both prior (PRE) and following (POST) a three-week static hip flexor stretching program. RESULTS: Following the stretching program, peak PHE increased significantly (P < 0.001), while no significant improvements were reported in AHE, APT, or LSE. In addition, no relationship was found between the change in PHE with either the change in AHE, APT, or LSE. Finally, a high relationship was observed between AHE and APT during running (r = 0.83, p < 0.001), and low relationships were observed between APT and LSE (r = -0.41, p = 0.08) and AHE and LSE (r = -0.34, p = 0.15). CONCLUSION: A three-week static stretching program of the hip flexor musculature resulted in an increase in PHE, but the sagittal plane kinematics of the LPH complex during running remained unchanged. The correlations observed between AHE, APT, and LSE suggest there is a kinematic relationship between the hip, pelvis, and spine.

Background/Purpose: Physical activity is commonly prescribed to reduce childhood obesity. However, due to differences in mechanics during low-impact activities, such as walking, obese children may be more prone to negative physical complications during high-impact activities, such as running. Therefore, this study analyzed the mechanical differences in running mechanics between healthy weight (HW) and overweight/obese (OV/OB) children. We hypothesized that when compared to HW children, OV/OB children would display higher vertical loading, greater joint moments and greater joint angular impulses during running. We also expect decreased sagittal plane range of motion and increased frontal plane range of motion of the hip, knee, and ankle joints in the OV/OB group during running. Methods: Ground reaction force (GRF) and joint kinematic data were collected for 42 children (25 HW, 17 OV/OB) while they ran across an implanted GRF platform at a given speed of 3.5 ± 5% m/s. Spatial-temporal and joint kinetic data (ankle, knee, & hip) were also determined. A one-way ANOVA was used to compare group differences for all variables of interest (p

Purpose: The purpose was to measure the effects of intermittent pneumatic compression (NormaTec, NT) on muscle inflammation after long distance running. Methods: Ten long distance runners, five males and five females, ages 18-55 years performed two, 20 mile runs at 70% VO2 max. The runs were followed by either no treatment (control) or NT treatment for five consecutive days. For the NT run, subjects were treated for one hour immediately following the run and daily for five days after. For the control run, subjects did not receive any treatment. Serum C – reactive protein (CRP), a marker of muscle inflammation, was measured pre and post run and daily thereafter for five days for both trials. Repeated measures ANOVA and two-way ANOVA were used to assess treatment differences. Results: The results indicated no significant difference (P > 0.05) between the control and treatment runs in CRP levels. There was also no gender differences or order effect of runs. Subjective pain ratings indicated no significant difference in pain between the control and treatment runs except when comparing the first run compared to the second run (regardless of whether the first run was a treatment or control run); there was a significant difference in which the subjects experienced less pain on the second run. There was no significant difference in mean weight loss, fluid intake, sweat rate, heart rate, percentage of maximum heart rate, or percentage of maximum VO2 max. There was, however, a significant difference (P = 0.038) in running time when comparing the control run (196.2 minutes) against the treatment run (204.8 minutes). Conclusions: Although the test subjects recovered one day earlier when using the NT device (Day 4) compared to the control (Day 5), this difference was not significant.

INTRODUCTION: Excessive hip adduction (HADD) has been associated with a number of lower extremity overuse injuries, and it has been suggested that it may be the result of reduced strength of the hip abduction musculature. Hip structure has been postulated to influence both hip abduction (HABD) strength and HADD. The purpose of this study was to investigate the relationship between hip structure, HABD strength, and frontal plane kinematics during running. METHODS: Peak isometric HABD strength, lower extremity kinematics, femoral neck-shaft angle (NSA), and pelvis width-femur length (pw-fl) ratio were recorded for 25 female subjects. Pearson correlations (P < .05) were performed between variables. RESULTS: A fair relationship was observed between femoral NSA and HABD strength (r = -.472 P = .017) where an increased NSA was associated with reduced HABD strength. No relationship was observed between HABD strength and frontal plane kinematics or between NSA/pw-fl and frontal plane kinematics. CONCLUSION: Alterations in the femoral NSA have the ability to influence peak isometric hip abduction strength. However, alterations in strength did not result in changes in lower extremity kinematics. Structural deviations at the hip do not appear to influence hip kinematics during running.

Objective: The purpose of this study was to investigate the effect of different orthotic shoe inserts on kinematics of the lower extremity and center of pressure (COP) during treadmill running. Design: The study design was an experimental investigation of five different orthotic insert conditions. Background: Orthotic shoe inserts have been used to treat a wide variety of running injuries. Despite their high success rate, the mode of action of orthotics is not well understood. Orthotics are often prescribed to reduce overpronation that has traditionally been evaluated through tibiocalcaneal eversion. The increased use of three-dimensional (3-D) kinematic methods in recent years has created an emphasis on tibia internal rotation as a component of subtalar joint pronation. No studies have yet shown a systematic effect of a series of medial and lateral heel postings on kinematics during running. Methods: The subtalar joint axes of seven healthy male subjects (mean age: 25.1 ± 2.3 yrs.) were measured, and the subjects performed treadmill running at 3. 8 m s-1 in a running shoe with the factory insoles (control) and four other orthotic conditions: 5 ° lateral post, neutral, 5 ° medial post, and 10° medial post. 3-D kinematic data of the foot and leg segments were captured using a four-camera system at 120 Hz. (Vicon), and COP data were collected with an in-shoe plantar pressure system at 120 Hz. (F-Scan). A oneway repeated measures ANOV A with post hoc comparisons was used to determine significant differences between conditions. Results: The orthotics had no significant effect on frontal plane kinematics. Tibia internal rotation was reduced with the use of a 5 ° medially posted orthotic. • The COP was shifted significantly posteriorly with increasing height of the heel. Conclusions: More studies are needed in order to relate anthropometrics and orthotics to predictable changes in kinematics.