A comprehensive assessment of the applicability of RoboColumn as a chromatography scale-down model for use in biopharmaceutical process validation.

High-throughput process development has become a standard practice in the biopharmaceutical industry to enable time, cost, and material savings. In downstream biopharmaceutical process development, miniaturized, parallelized chromatography columns, known as RoboColumn, have become the standard for process development, as RoboColumn have shown generally comparable performance to bench and manufacturing scale columns. However, RoboColumn have yet to be widely implemented in process validation and characterization, where many multifactor experiments are typically executed, and there is a strong value proposition for performing high-throughput experiments. The hesitancy to utilize RoboColumn in process validation arises from scale differences that result in exacerbated peak broadening at RoboColumn scale relative to traditional bench or manufacturing scales. Thus, to support reliable application of RoboColumn in process validation, the present study provides a comprehensive investigation to understand how scale differences affect chromatographic performance by comparing RoboColumn, bench, and manufacturing scales using seven different production processes covering three different antibody formats, five different resin types, and three chromatographic modes of operation. RoboColumn chromatographic performance was compared at target and off-target conditions to emulate scale-down model qualification and multifactor studies, respectively. RoboColumn demonstrated good comparability at both target and off-target process conditions. To further demonstrate an understanding of comparability, a study was performed to show a rare case in which product quality offsets may occur as a result RoboColumn scale differences. By showing scale comparability and an understanding of potential offsets, this work demonstrates that RoboColumn can be used in any stage of process development, including process validation and characterization.

Predicting Formulation Conditions During Ultrafiltration and Dilution to Drug Substance Using a Donnan Model with Homology-Model Based Protein Charge.

In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.

Simplified strategy for developing purification processes for antibody-drug conjugates using cation-exchange chromatography in flow-through mode.

The development of manufacturing processes for antibody-drug conjugates (ADCs) presents many challenges compared to a standard monoclonal antibody. Conjugation processes typically start with an antibody intermediate that was purified to have very low levels of aggregates. The additional processing required for ADCs, including a hydrophobic small molecule and co-solvents, contributes to unacceptable levels of protein aggregate species. A post-conjugation purification step could be necessary to ensure that the process robustly delivers a product that achieves the desired product quality specifications. This paper describes a methodology for developing chromatographic purification steps to remove very high molecular weight species (vHMWS) in ADCs and was applied to three products, decreasing the vHMWS by ≥ 85% to ≤ 0.1%. Leveraging the antibody intermediate purification conditions to streamline development, we efficiently developed robust flow-through cation-exchange chromatography steps for ADC products.

Characterizing and enhancing virus removal by protein A chromatography.

Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal.

Objective Evaluation of the Effects of Deployment on Laparoscopic Skills: The Simulation and Deployment Laparoscopic Skills Study.

OBJECTIVE: To assess the effects of deployment on basic laparoscopic skills of general surgeons and obstetrics/gynecology (OB/GYN) physicians. METHODS: This was a prospective 10-site study. Active duty Army OB/GYN and general surgery physicians scheduled to deploy were invited to participate. Before deployment, they performed fundamentals of laparoscopic surgery (FLS) tasks and specialty-specific procedures on a virtual reality laparoscopic simulator. Upon returning, physicians repeated the same evaluations. Questions about perceived comfort with laparoscopic procedures were asked before and after deployment. Statistical analysis included paired t tests for continuous variables and nonparametric for ordinal data with a p value of <0.05 considered significant. RESULTS: 121 deploying providers were invited to participate; 35 agreed and 29 completed the predeployment skills assessment. After deployment, 15 providers had postassessment evaluation data collected, and their results were used for analysis. Though physicians reported a decrease in their perception of preparedness for advanced laparoscopic procedures and complications, there was no decrement in their performance of FLS tasks or the basic laparoscopic procedures. CONCLUSION: Time away from regular clinical practice during deployments did not significantly affect surgeons' performance as measured by a virtual reality laparoscopic simulator. Additional study on effects on advanced procedures should be considered.

Self-Reported Changes in Comfort Level With Basic OB/GYN Procedures After Deployment.

OBJECTIVE: To assess the specialty-specific procedures and clinical encounters U.S. Army obstetrician/gynecologist (OB/GYN) providers felt were affected by deployment and identify skills and areas that may benefit from postdeployment training. METHODS: Active duty Army OB/GYN physicians were invited to participate in an anonymous web-based survey to rate their comfort level and experience performing specialty-specific procedures before and after military deployment. Physicians rated their comfort level on a 5-point Likert scale (1 = very uncomfortable to 5 = completely comfortable). Statistical analysis included Pearson χ(2) and McNemar's χ(2) with a p value of <0.05 considered significant. RESULTS: Of the 100 physicians eligible to complete the survey, 66 responded (response rate = 66%). Their responses demonstrated a statistically significant perception of decline in their comfort level with nearly every obstetric and gynecologic procedure and clinical encounter evaluated. CONCLUSION: Time away from regular clinical practice during deployment results in providers feeling less comfortable with many common OB/GYN procedures and clinical situations. Although these results are not a measure of actual performance, this information helps define targets to focus refresher training for providers who leave their normal scope of practice for deployment. These findings apply to civilian providers leaving practice for other reasons as well.

Pipelle endometrial sampling in the evaluation of abnormal first trimester pregnancies.

OBJECTIVE: To evaluate the utility of pipelle endometrial sampling as a diagnostic tool in the evaluation of abnormal first trimester gestations. METHODS: Thirty-one women with abnormal first trimester pregnancies defined as gestations with abnormally rising or falling quantitative human chorionic gonadotropin (ß-hCG) levels and ultrasound findings consistent with a nonviable or ectopic pregnancy were prospectively evaluated. Endometrial pipelle sampling was performed on each patient in a standardized fashion immediately before curettage and evaluated as a fixed, permanent specimen to assess for the presence of either chorionic villi, trophoblastic or fetal tissue. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value for endometrial biopsy were 70.1%, 100%, 100%, and 33.3%, respectively. For curettage, the sensitivity, specificity, positive predictive value, and negative predictive value were 88.9%, 100%, 100%, and 57.1%, respectively. CONCLUSION: Curettage's overall sensitivity is superior to endometrial biopsy in detecting the presence of intrauterine products of conception in early abnormal pregnancies. However, in some clinical situations where the diagnosis of ectopic pregnancy is in question, particularly if the ß-hCG is ≤ 2,000 mIU/mL, endometrial pipelle biopsy could be a useful tool in the evaluation algorithm.

Practice and perceptions of obstetrics and gynecology providers and the treatment of overweight and obese women.

OBJECTIVE: To verify how frequently obstetrics-gynecology providers documented the diagnosis and treatment of overweight/obese patients during routine examinations. These results were compared to a survey administered to this group of providers that measured their perceived practice patterns regarding weight management. STUDY DESIGN: A retrospective chart review was performed of well woman and routine postpartum exams of patients with a body mass index (BMI) > 25. Patient encounters were reviewed for a diagnosis of overweight or obese and weight management interventions offered. In addition, a survey was administered to obstetrics-gynecology providers who performed these exams in order to measure their perceived obesity diagnosis and treatment patterns. RESULTS: A total of 1,145 patient encounters were reviewed of patients with a BMI > 25. Providers documented 77 of the 1,145 (6.7%) as overweight or obese. Only 70 of 1,145 (6.1%) had documented interventions (i.e., recommending weight loss or exercise). In contrast, a majority of providers surveyed reported they appropriately diagnosed and counseled patients with obesity. CONCLUSION: The majority of obstetrics and gynecology providers surveyed perceived that weight management was an important part of their clinical practice; however documentation of patient encounters did not match their perceived clinical

Use of a postpartum hemorrhage simulator for instruction and evaluation of residents.

OBJECTIVE: Postpartum hemorrhage is a common and potentially life-threatening obstetric emergency. We sought to create a realistic simulation and validate a standardized grading form to evaluate competency in the management of postpartum hemorrhage. METHODS: Residents from 3 programs underwent training with a postpartum hemorrhage simulation using a standard obstetric birthing model equipped with an inflatable uterus to simulate uterine atony. All simulations were graded by staff physicians with a standardized grading sheet constructed from the current literature on the topic. Residents were expected to recognize the hemorrhage and take appropriate steps, including asking the assistant to administer medications, to correct the problem. Objective and subjective performance was measured with standardized grading sheets, and results were analyzed for reliability using Cronbach α and intraclass correlation coefficients. This project was conducted in accordance with the hospital Institutional Review Board policies at each institution. RESULTS: Forty residents from 3 institutions underwent simulation training. The majority were unable to correct the hemorrhage within 5 minutes and almost half also made at least 1 error, either the dose or route, in the medications they requested. Reliability was evaluated with Cronbach α and demonstrated the grading sheets were valid and had good interrater reliability. DISCUSSION: A simulated postpartum hemorrhage scenario can identify important deficiencies in resident knowledge and performance, with no risk to patients. The standardized grading form worked well for our purposes and was reliable in our study. Further testing is needed to evaluate whether the training improves performance in real-life hemorrhages.

A curriculum for Burch colposuspension and diagnostic cystoscopy evaluated by an objective structured assessment of technical skills.

OBJECTIVE: The purpose of this study was to develop an effective curriculum for teaching colposuspension and diagnostic cystoscopy. STUDY DESIGN: Fifty-five residents underwent an Objective Structured Assessment of Technical Skills that was composed of a task-specific checklist and validated global assessment. Thirty of the residents had been exposed to a training curriculum, and 25 residents served as untrained control subjects. RESULTS: For the colposuspension and cystoscopy checklists, the reliability coefficient was 0.85 and 0.72, and the interrater reliability was 0.92 and 0.68, respectively. Although residents who were provided the curriculum performed better on both task-specific checklists, the differences did not reach statistical significance. Senior residents performed consistently better than junior residents for both tasks. When a comparison was made of the junior residents separately to account for previous experience, trained residents performed significantly better on the cystoscopy checklist (P = .029). CONCLUSION: This curriculum is an effective way to teach diagnostic cystoscopy to junior residents. The checklist for this Objective Structured Assessment of Technical Skills has good reliability and construct validity.

Comparison of objective, structured assessment of technical skills with a virtual reality hysteroscopy trainer and standard latex hysteroscopy model.

OBJECTIVE: To evaluate a virtual reality (VR) hysteroscopy device and a standard latex hysteroscopy model for assessment of residents' surgical skills. STUDY DESIGN: As part of a 7-station, objective, structured assessment of technical skills (OSATS), 13 obstetrics and gynecology residents performed myoma resection with the Immersion Hysteroscopy VR Trainer (Gaithersburg, Maryland) and dissection of a polyp with a model from Limbs and Things, Inc. (LT) (Bristol, England). OSATS were scored by both blinded (to the residents' levels) and unblinded examiners using a task-specific checklist, global rating scale and pass/fail assessment. RESULTS: Reliability indices for the VR model were 0.70 (checklist) and 0.98 (global), with an interrater reliability of 0.87 and 0.92, respectively. Correlation of scores between blinded and unblinded examiners was 0.78 for the checklist and 0.88 for the global (p < 0.001). Examination of construct validity revealed improved scores with-increasing postgraduate year. Comparison of VR scores to LT scores revealed VR scores to be significantly higher than LT scores (p < 0.001). Correlations of VR and LT scores were low for both the global (0.478, p = 0.12) and checklist (0.362, p = 0.24). CONCLUSION: The VR Hysteroscopy Trainer is not superior to standard models for evaluating surgical skills.

A new curriculum for hysteroscopy training as demonstrated by an objective structured assessment of technical skills (OSATS).

OBJECTIVE: The purpose of this study was to develop an effective curriculum to teach hysteroscopy, as well as to develop an objective assessment instrument (OSATS). STUDY DESIGN: All 24 residents in our training program attended a 3-hour hysteroscopy curriculum in each of 3 years. Each year after the curriculum, an OSATS was administered consisting of an assembly and operative task. Each task was evaluated with a task-specific checklist and a previously validated global assessment form. Twenty-four residents from an outside institution served as controls. All residents were examined by blinded and unblinded examiners. RESULTS: The reliability coefficient was .7857 for the checklist and .9763 for the global scale. Interrater reliability for the checklist was .7478 and ranged from .4222 to .9329 for the global instruments. Evaluation of construct validity revealed that the junior residents at both locations did significantly worse on the checklist and global scale than the senior residents for all 3 years of the study (P < .001). Those residents provided the curriculum did significantly better on both the checklist and the global scale than the resident controls with a mean of 45% versus 63% for the checklist and 18.3 versus 24.9 for the global score. (P = .001 checklist, P = .007 global scale). CONCLUSION: This curriculum is an effective way to impart knowledge and skill in the assembly and use of the operative hysteroscope. The checklist developed for this OSATS has excellent reliability and construct validity.