Implementation of QbD for the development of a vaccine candidate.

This case study provides an example of how Quality by Design (QbD) principles were applied to accelerate process development to manufacture a vaccine candidate at commercial scale. By leveraging an existing manufacturing platform process, a risk assessment was used to differentiate process parameters that could be defined using a combination of scientific and historical manufacturing knowledge from those that merited additional process characterization by experimentation. Select parameters, and their interactions, were evaluated by a Design of Experiment (DoE) series. This systematic approach required less time and fewer resources and resulted in the definition of a reliable and robust manufacturing process that meets regulatory requirements.

Preparation-free method for detecting Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef particulates.

We show the detection of 100 cells per ml of Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef washes and particulate matter with piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. The PEMC sensors (sensing area, 2 mm2) were immobilized with polyclonal antibody specific to E. coli O157:H7 (EC) and were exposed to 10 aqueous washes of locally purchased spinach, spring lettuce mix, and ground beef for testing if EC was present. Absence of resonance frequency shift indicated that EC was not present in the 30 samples tested. Following the last sample in each food matrix, 1,000 cells per ml of EC were spiked into the sample container, and resonance frequency change was monitored. The total resonance frequency change was 880 +/- 5, 1,875 +/- 8, and 1,417 +/- 4 Hz for spinach, spring lettuce mix, and ground beef, respectively. A mixture of the three food matrices spiked with 100 cells per ml of EC gave a sensor response of 260 +/- 15 Hz. The resonance frequency changes are approximately 40% lower than our previously reported study on ground beef. It is suggested that the reduction in sensitivity is due to differences in pathogen adherence to food matrices, which affects target binding to the sensor surface. We conclude that detection selectivity is conserved in the three food matrices examined and that the magnitude of sensor response is a function of the food matrix.

Detection and confirmation of staphylococcal enterotoxin B in apple juice and milk using piezoelectric-excited millimeter-sized cantilever sensors at 2.5 fg/mL.

A sensitive and reliable method for the detection of a model toxin, staphylococcal enterotoxin B (SEB) in buffer, apple juice, and milk is shown using piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors. Limit of detection in spiked milk and apple juice samples is 10 and 100 fg, respectively. PEMC sensors (2 mm(2)) are prepared by immobilizing a polyclonal antibody specific to SEB, which was exposed to 1 mL of 1% milk and apple juice containing 10 fg-10 ng. Sensor response to 100 fg, 1 pg, and 10 pg of SEB in apple juice resulted in resonance frequency decreases of 113 +/- 18 (n = 4), 308 +/- 24 (n = 4), and 521 +/- 20 (n = 2) Hz, respectively. In milk, 10 fg, 100 fg, 1 pg, and 10 pg of SEB resulted in resonance frequency decreases of 126 +/- 18 (n = 2), 143 +/- 35 (n = 4), 310 +/- 32 (n = 5), and 557 +/- 25 (n = 2) Hz, respectively. Positive detection of SEB in the sample solution was observed within the first 20 min. The responses of the sensor to positive (SEB present, but no antibody on sensor), negative (SEB absent, antibody on sensor), and buffer (SEB absent, antibody on sensor) controls were -17 +/- 10 (n = 3), -9 +/- 5 (n = 3), and -6 +/- 12 (n = 18) Hz, respectively. Positive verification of SEB detection was confirmed by two methods: (1) low-pH buffer release caused increase in resonance frequency, and (2) second antibody binding to SEB attached to sensor that caused further resonance frequency decrease. The significance of these results is that PEMC sensors can reliably detect SEB at 10-100 fg (effective concentration of 2.5 and 25 fg/mL) in complex fluids without sample preparation or the use of labeled reagents.

Method for quantification of a prostate cancer biomarker in urine without sample preparation.

We describe a macrocantilever-based method for detecting a prostate cancer biomarker (alpha-methylacyl-CoA racemase; AMACR) directly in patient urine without a sample preparation step and without the use of labeled reagents. Clean catch voided urine specimens were prospectively collected from five confirmed prostate cancer patients 3 weeks postbiopsy. The presence of AMACR was measured in a blinded manner by exposing 3 mL of urine to the anti-AMACR-immobilized piezoelectric-excited millimeter-sized (PEMC) sensor. The resonance frequency of PEMC decreases as AMACR from sample binds to the antibody on the sensor. The resonance frequency changes for the five patients tested were 4,314 +/- 35 (n = 2), 269 +/- 17 (n = 2), 977 +/- 64 (n = 3), 600 +/- 31 (n = 2), and 801 +/- 81 (n = 2) Hz, respectively. Positive detection was observed within approximately 15 min. The responses to positive, negative, and buffer controls were -9 +/- 13, -34 +/- 18, and -6 +/- 18 Hz, respectively. Positive verification of AMACR attachment was confirmed by low-pH buffer release. The sensor response was quantitatively related to AMACR concentration in control urine, and the relationship was used in developing an in situ calibration method for quantifying AMACR in patient urine. Estimated concentrations of 42, 2, and 3 fg/mL AMACR were calculated for the three patients' urine, while absence of AMACR was confirmed in control urine (n = 13). Because of simplicity of measurement combined with high sensitivity and specificity, the method may be a useful adjunct in a point-of-care setting to identify men at increased risk for prostate cancer.

10-minute assay for detecting Escherichia coli O157:H7 in ground beef samples using piezoelectric-excited millimeter-size cantilever sensors.

We detected Escherichia coli O157:H7 (EC) at approximately 10 cells per ml in spiked ground beef samples in 10 min using piezoelectric-excited millimeter-size cantilever (PEMC) sensors. The composite PEMC sensors have a sensing area of 2 mm2 and are prepared by immobilizing a polyclonal antibody specific to EC on the sensing surface. Ground beef (2.5 g) was spiked with EC at 10 to 10,000 cells per ml in phosphate-buffered saline (PBS). One milliliter of supernatant was removed from the blended samples and used to perform the detection experiments. The total resonant frequency change obtained for the inoculated samples was 138 +/- 9, 735 +/- 23, 2,603 +/- 51, and 7,184 +/- 606 Hz, corresponding to EC concentrations of 10, 100, 1,000, and 10,000 cells per ml, respectively. EC was detected in the sample solution within the first 10 min. The responses of the sensor to positive, negative, and buffer controls were 36 +/- 6, 27 +/- 2, and 2 +/- 7 Hz, respectively. Verification of EC attachment was confirmed by low-pH buffer release (PBS-HCl, pH 2.2), microscopy, and second antibody EC binding postdetection. The results indicate that PEMC sensors can reliably detect EC at less than 10 cells per ml in 10 min without sample preparation and with label-free reagents.

Method for label-free detection of femtogram quantities of biologics in flowing liquid samples.

Rapid (approximately 10 min) measurement of very low concentration of pathogens (approximately 10 cells/mL) and protein (approximately fg/mL) has widespread use in medical diagnostics, monitoring biothreat agents, and in a broader context as a research method. For low-level pathogen, we currently use culture enrichment methods and, thus, rapid analysis is not possible. For low protein concentration, no direct method is currently available. We report here a novel macrocantilever design whose high-order resonant mode near 1 MHz exhibits mass detection sensitivity of 10 cells/mL for cells and 100 fg/mL for protein. The sensor is 1x3 mm and uses a piezoelectric layer for both actuation and sensing resonance. Sample is flowed (approximately 1 mL/min) past the antibody-immobilized sensor, and as antigen binds to the sensor, resonance frequency decreases in proportion to antigen concentration. The sensor showed selectivity to the pathogen even though copious nonpathogenic variant was simultaneously present.

Measuring bacterial growth by tapered fiber and changes in evanescent field.

Single mode continuous tapered fibers were fabricated with waist diameters of 6-8 microm and of 11 mm waist lengths. The tapered surface was coated with poly-l-lysine and Escherichia coli (E. coli) (JM 101) expressing green fluorescent protein was immobilized. Growth of this culture at 22 and 32 degrees C was monitored by 480 nm light transmission through the tapered fiber. Change in transmission is a measure of change in absorption of the evanescent field. The transmission decreased exponentially with cell growth on the tapered surface. Growth rate was determined and compared favorably with cells grown on the same medium in multiwell plates. Significance of the results is that a tapered fiber sensor can be used effectively for rapid assessment to determine the presence of bacteria by growth.