Validation of an automated method for compounding monoclonal antibody patient doses

Automation robots have recently come to the market as an alternative for manual compounding of drugs for intravenous administration. Our aim was to assess whether robotic compounding can be performed with monoclonal antibodies (mAbs) without influencing the aggregation state of the proteins. Three frequently used mAbs were studied: infliximab (Remicade^®, Janssen Biotech) and trastuzumab (Herceptin^®, Roche) in lyophilised form, and bevacizumab (Avastin^®, Roche) as a liquid formulation stored at 2°C to 8°C. The effects of different procedures to prepare the patient doses on antibody aggregation were evaluated. Remicade^® and Herceptin^® were reconstituted both manually and by a robotic arm (i.v.STATION^®, Health Robotics). Additionally, the influence of vigorous shaking during reconstitution was investigated. The effects of rapid aspiration and dispensing on antibody aggregation were investigated for all three mAbs. Aggregation state was assessed by UV-Vis absorbance, 90° light scatter, fluorescence spectroscopy, Nile red fluorescence microscopy, and field flow fractionation without cross and focus flow. Robotic reconstituted samples showed similar findings compared with manual reconstitution if performed exactly according to the summary of product characteristics (SPC). Vials that were vigorously shaken showed a significant increase in aggregates. Similarly, rapid aspiration/dispense cycles resulted in a strong increase in the number and sizes of aggregates for all three mAbs; this result was observed after just one rapid aspiration/dispense cycle. Our study showed that robotic compounding of mAbs is feasible if the robot is exactly programmed according to the SPC, indicating that robotic compounding can be used to achieve reproducible high-quality compounding for delicate formulations.     

Statistical Series: Opportunities and challenges of sperm motility subpopulation analysis

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Cloning, expression, purification and functional characterization of recombinant human PSP94

Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS-PAGE analysis and a peak with a molecular mass (M(r)) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, in vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.     

Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa

Viable epididymal sperm can be obtained in the Spanish ibex during 24h after death, but it has been observed a significant effect of the post-mortem time on fertility success, so only goats inseminated with semen recovery during the first 8h became pregnant. The aim of this study was to determine the effect of post-mortem time on epididymal semen samples from of Spanish ibex. For this purpose, sperm samples from 36 males were collected at different post-mortem times, from 2 to 24h, and cryopreserved. Thawed samples were incubated for 2h at 37°C without dilution or after dilution in a modified Tyrode medium, in order to study the sperm resistance to dilution. Moreover, flow cytometry was used to assess the sperm viability (PI), phospolipid disorder of the plasma membrane (M540), mitochondrial membrane potential (Mitotracker Deep Red), indirect apoptosis markers (YOPRO-1) and sperm chromatin stability (SCSA(?)). Sperm motility was evaluated by computer-assisted sperm analysis (CASA). Our results have shown that post-mortem time caused a reduction in mitochondrial membrane potential. In this regard, the loss of energy could be responsible for the loss of maintenance of the membrane with a consequent increase in permeability leading to a decrease in sperm viability and motility, losing linearity and speed. Moreover, the loss of maintenance of the membrane influence the extent to which sperm will survive the cryopreservation process, as it shows the results obtained from the dilution-incubation resistance test. Finally, one important finding of this study is the demonstration of no effect of post-mortem time on post-thaw DNA integrity, giving us the possibility of using sperm samples from valuable males, even if it was not possible to process during the first 8h.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility,viability, and morphological integrity

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.