Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing.

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Pathways From Witnessing Parental Intimate Partner Violence to Involvement in Bullying: Empirically Testing a Proposed Conceptual Framework.

We tested a proposed conceptual framework in which we hypothesized that witnessing parental intimate partner violence (witnessing parental IPV) is linked to children's bullying and peer victimization. We also hypothesized that the relationship between witnessing parental IPV and bullying and peer victimization in childhood would be mediated by increased psychological problem behavior, school absences, and problematic peer interactions. We utilized data from the National Survey of Children's Health. We found that witnessing parental IPV was positively related to children's bullying and peer victimization not only directly, but also indirectly through the mediating role of psychological problem behavior, school absences, and problematic peer interactions. Our findings highlight the importance of exploring the mechanisms by which bullying and peer victimization increase in those who have witnessed parental IPV.

Determinants of intergenerational sexual relationships and HIV risk among South African women outpatients in Gauteng.

Age-discrepant sexual relationships may place women at risk for HIV infection in South Africa. Results are based on medical records and case-control interview data of 889 sexually experienced women outpatients aged 15-29 in a Gauteng township. Women with partners at least ten years older (intergenerational) show an elevated chance of having an early sexual debut, concurrent and transactional partners, and intimate partner violence. Hardship during childhood including parental loss, food insecurity and abuse are related to age asymmetric relationships. HIV is two times more likely (aOR=1.96) with an intergenerational partner. Transactional sex increases the odds of HIV independently (aOR=1.76) as does intimate partner violence (aOR=1.6). To the extent that transactional and intergenerational sex overlap the chance of contracting HIV increases more than two-fold (uOR=4.57). Girls (15-19) with intergenerational partners face the highest chance of HIV (uOR=8.55) compared to other age groups. They are also five times more likely than controls to have lived with a cross-age partner. Our findings indicate there are multiple pathways emanating from childhood leading to women's choice of intergenerational partners, and the link to HIV is strongest among the youngest women.

The role of reproductive coercion in women's risk for HIV: A case-control study of outpatients in Gauteng, South Africa.

OBJECTIVES: To investigate how reproductive coercion, or men's attempts to control their partners' use of contraception, may contribute to adverse reproductive health outcomes for women including abortions, sexually transmitted infections, and HIV for young women in South Africa. METHODS: Findings are based on a case-control interview study of 882 South African women outpatients aged 15-29 years, 48.5% (n=427) of whom were HIV seropositive. Covariates include demographics, intimate partner violence, sexually transmitted infections, having an abortion, using long-acting reversible contraception, and unequal sexual relationship power. RESULTS: Most covariates with the exceptions of abortion and unequal relationship power increase the risk of HIV, and all relate to reproductive coercion. Intimate partner violence is strongly associated with reproductive coercion (odds ratio 3.86, 95% confidence interval 2.89-5.15). When intimate partner violence is included in the full model reproductive coercion remains a significant predictor of HIV by 42%, and acts as a partial mediator between IPV and HIV. CONCLUSION: Findings confirm the significance of reproductive coercion as a risk marker for HIV. Reproductive coercion undermines women's reproductive health and warrants clinical intervention. Recommendations are offered for clinical practice within the South African context to increase training and assessment and provide covert long-acting reversible contraception as one pathway towards promoting women's reproductive autonomy.

Safety and Efficacy of OXB-202, a Genetically Engineered Tissue Therapy for the Prevention of Rejection in High-Risk Corneal Transplant Patients.

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye, corneal transplantation is one of the most successful clinical transplant procedures. However, in high-risk patients, which account for >20% of the 180,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient cornea. The main reason for graft failure is irreversible immunological rejection, and it is therefore unsurprising that neovascularization (NV; both pre and post grafting) is a significant risk factor for subsequent graft failure. NV is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the equine infectious anemia virus called pONYK1EiA, which subsequently prevents rejection by suppressing NV. Previously, it has been shown that rabbit donor corneas treated with pONYK1EiA substantially suppress corneal NV, opacity, and subsequent rejection in an aggressive rabbit model of cornea graft rejection. Here, efficacy data are presented in a second rabbit model, which more closely mirrors the clinical setting for high-risk corneal transplant patients, and safety data from a 3-month good laboratory practice toxicology and biodistribution study of pONYK1EiA-modified rabbit corneas in a rabbit corneal transplant model. It is shown that pONYK1EiA-modified rabbit corneas (OXB-202) significantly reduce corneal NV and the rate of corneal rejection in a dose-dependent fashion, and are tolerated with no adverse toxicological findings or significant biodistribution up to 13 weeks post surgery in these rabbit studies. In conclusion, angiogenesis is a valid target to prevent corneal graft rejection in a high-risk setting, and transplanted genetically modified corneas are safe and well-tolerated in an animal model. These data support the evaluation of OXB-202 in a first-in-human trial.

Estimation of hematocrit in filter paper dried bloodspots by potassium measurement: advantage of use of perimeter ring samples over circular center sub-punch samples.

BACKGROUND: Quantitative assays using dried filter paper bloodspots (DBS) may be adversely affected by hematocrit (HCT) as an unknown variable. Studies have demonstrated the utility of the measurement of potassium (K) from DBS punches to estimate HCT. Because there is significant accrual of RBCs at the DBS perimeter, we investigated whether K measurement from ring-shaped specimens inclusive of the perimeter might provide an advantage over conventional interior circular sub-punch samples to estimate HCT. METHODS: Primary samples were Li-heparin whole blood, with HCT as measured on concurrently-drawn K-EDTA specimens. DBS were formed by bolus addition of 40 µL whole blood to filter paper cards. Total bloodspot area was determined by image analysis. Removal of center sub-punch (P) samples of fixed area produced remainder ring (R) samples inclusive of the perimeter. Samples were extracted in K-EDTA (2.5 mmol/L) and measured for diluent-corrected K per area (α, µmol K/cm2). RESULTS: Forty-three patient samples were utilized. α was normally distributed: α(P)=1.23±0.26 µmol K/cm2; α(R)=1.86±0.41 µmol K/cm2; α(R)/α(P)=1.51±0.15. α was correlated with HCT: α(P)=0.030 HCT(%)+0.015 µmol K/cm2 (r2=0.795); α(R)=0.052 HCT(%)+0.010 µmol K/cm2 (r2 = 0.912), but with higher resolution and lesser error for α(R). CONCLUSIONS: K per area (α) was significantly higher in R samples vs. P samples, with higher resolution for α(R) vs. HCT. Use of ring samples inclusive of the perimeter to estimate HCT for DBS via K measurement can provide an advantage over use of center sub-punch samples.

Intimate Partner Violence is Associated with Voluntary Sterilization in Women.

BACKGROUND: Intimate partner violence (IPV) may interfere with women's use of preferred forms of contraception, resulting in unwanted pregnancies forcing women to seek permanent sterilization. A history of child sexual abuse (CSA) presages the risk for IPV in adulthood setting the stage for adverse reproductive outcomes. OBJECTIVE: To determine whether CSA and IPV are associated with women's voluntary sterilization when adjusting for demographics and reproductive health history. METHODS: This cross-sectional study is based on in-person interviews of women (N = 278) drawn from outpatients surveyed in more than 10 different clinics (N = 2465). Women's history of gender-based violence and bilateral tubal ligation (BTL) were assessed. RESULTS: About half of the women had a past history of IPV and 29% disclosed CSA. CSA predicted later entry into an abusive relationship (odds ratio [OR] = 6.7). Sterilization was reported by 19.6%. Parity (3+ children), having had an abortion, and receipt of welfare were associated with sterilization in univariate tests. Among those women receiving a BTL, 74% had violent partners. Adjusted multivariate logistic regressions, adjusted for demographics and reproductive history, indicated that having had an abusive partner increased the odds of sterilization; parity was also highly associated. CSA exerted only an indirect influence on sterilization via entry into violent relationships. CONCLUSION: IPV raises the likelihood that women will choose sterilization. Despite the importance of women's access to permanent contraception, priority should be given to screening for gender-based violence and promoting interventions.

Optimizing Transgene Configuration and Protein Fusions to Maximize Dopamine Production for the Gene Therapy of Parkinson's Disease.

Pharmacological dopamine replacement therapies provide the most well-established treatments for Parkinson's disease (PD). However, these long-term treatments can lead to motor complications and off-target effects. ProSavin(®), a lentiviral vector (LV)-based gene therapy approach aimed at restoring local and continuous dopamine production, through delivery of three enzymes in the dopamine biosynthesis pathway, was demonstrated to be safe and well-tolerated in a phase I/II clinical study of patients with advanced PD. Although improvements in motor behaviour were observed, the data indicated that higher levels of dopamine replacement might be required to maximize benefit. We attempted to increase production of dopamine, and its precursor L-Dopa in LV-transduced cells, by optimizing the gene order in the ProSavin expression cassette, and by creating fusions of two or three of the transgenes, using linker sequences. In vitro analysis showed that several gene arrangements provided significantly increased dopamine and/or L-Dopa production compared with ProSavin, and that LV titers and transgene expression were not affected by introducing gene fusions. One vector, equine infectious anemia virus (EIAV)-TCiA, was selected for further characterization and showed significant improvements in dopamine and L-Dopa production compared with ProSavin, in human neuronal cells. Further characterization of EIAV-TCiA demonstrated expression of all three dopamine enzymes in vivo and faithful delivery and integration of the expected gene expression cassette within the genome of target cells, as assessed by Northern and Southern blotting. In conclusion, we have developed a novel LV vector with an increased capacity for L-Dopa and dopamine production compared with the current ProSavin vector. Clinical evaluation of this vector will be performed to assess the benefits in patients with PD.

The Effects of Gender-based Violence on Women's Unwanted Pregnancy and Abortion.

The aim of this research is to understand how gender-based violence across the life-course affects the likelihood of abortion. Women outpatients (n = 309) revealed their exposure to four different forms of gender-based abuse: child sexual abuse (25.7 percent), teenage physical dating violence (40.8 percent), intimate partner violence (43.1 percent), and sexual assault outside an intimate relationship (22 percent). Logistic regressions revealed that no single form of gender-based abuse predicted abortion. The cumulative effect of multiple forms of abuse did increase the odds of having an abortion (OR = 1.39, CI = 1.13-1.69). Child sexual abuse predicted intimate partner violence (OR = 6.71, CI = 3.36-13.41). The cumulative effect of gender-based violence on women's reproductive health warrants further research. Priority should be given to screening for multiple forms of victimization in reproductive healthcare settings.

Model analysis of effect of canagliflozin (Invokana), a sodium-glucose cotransporter 2 inhibitor, to alter plasma 1,5-anhydroglucitol.

BACKGROUND: Renal reabsorption of 1,5-anhydroglucitol (AG) is competitively inhibited by elevated glucose and leads to depleted plasma AG in diabetes. Plasma AG recovery in diabetes normally correlates with improved glycemic control. However, use of sodium-glucose co-transporter 2 (SGLT2) inhibitors (e.g., canagliflozin) to treat diabetes by inhibition of renal glucose reabsorption can negate this correlation, via an indirect effect (increase of renal filtrate glucose concentration) to inhibit AG reabsorption by sodium-glucose co-transporter 4 (SGLT4). Conversely, then, AG measurement might be useful as an independent marker for SGLT2 inhibitor activity. METHODS: Using an AG mass balance model, we analyzed literature data on plasma AG before and after initiation of canagliflozin therapy (CT) to quantitatively characterize the effect of CT on AG reabsorption. RESULTS: According to model calculations, modest decreases (<5%) in fractional reabsorption of AG account for the drastic decrease in [AG] observed during CT. Decreases are predicted to be rapid (t1/2<3days) after CT initiation. CONCLUSION: CT negates the usual premise of AG measurement (that [AG] should increase with improved glycemic control). However, according to model calculations, a substantial and likely rapid effect of CT on [AG] means that AG measurement might provide an early marker for CT activity.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Interpatient distributions of bloodspot area per fixed volume of application: comparison between filter paper and non-cellulose dried matrix spotting cards.

BACKGROUND: Non-cellulose dried matrix spotting (DMS) cards are an alternative to filter paper (FP) for bloodspots. We compared the interpatient distributions of bloodspot areas between DMS and FP for a fixed volume of application of whole blood, and examined correlations of areas with hematocrit. METHODS: EDTA-whole blood adult patient samples (n=49; 25 males, 24 females) were utilized after routine measurement of hemoglobin and hematocrit. Replicate (4×) bloodspots were produced by bolus drop application of 50µL whole blood via a fixed-volume pipettor to either FP or DMS. Dried bloodspot areas were determined by image analysis. RESULTS: Hematocrits (HCT) were normally distributed (HCT=30.9±5.3%). For both FP and DMS, bloodspot areas (a, cm(2)) across patients were normally distributed: for FP, a=1.11±0.056cm(2) (±5.0%); for DMS, a=0.378±0.037cm(2) (±9.9%). Relative bloodspot area differences across the population range were >20% for both DMS and FP. Correlation of bloodspot areas to hematocrit was negative for FP (r=-0.80) but positive for DMS (r=+0.78). CONCLUSIONS: Interpatient variation in blood volume per area is a preanalytical variable for both DMS and FP bloodspots. Hematocrit is but one interpatient variable, as correlations of fixed-volume bloodspot areas with hematocrit across patients were substantially inexact (r(2)<0.65).

The intergenerational transfer of mother-daughter risk for gender-based abuse.

In this 10-year longitudinal study 150 mother-daughter pairs were recruited to participate in a study examining gender-based abuse across three generations. Forms of gender-based abuse included: child sexual abuse, witnessing intimate partner violence against their mothers, and intimate partner violence or dating violence in adolescence or adulthood. Daughters were interviewed when they were on average 9, 14, and 16 years old. Regression analyses revealed that if the grandmother (G1) was abused by her husband, her daughter (G2) was more likely to be sexually molested in childhood and was also more likely to be in an abusive relationship as an adult. If the mother (G2) was sexually abused as a child her daughter (G3) was at increased risk for child sexual abuse. In turn, child sexual abuse for the daughters related to their reports of dating violence in adolescence. Daughters (G3) who were sexually abused expressed more anxiety about romantic relationships, reflecting early attachment conflicts. Both child sexual abuse and anxious romantic attachment style independently predicted adolescent sexual risk-taking as in having multiple sexual partners or dating older men. These findings demonstrate how informative it is to include multiple forms of gender-based abuse in research and practice to better illuminate complex family dynamics. In addition, the findings support previous empirical work showing the importance of attachment behavior in women who are in abusive relationships, which has unique clinical implications.

Characterization of lentiviral vector production using microwell suspension cultures of HEK293T-derived producer cells.

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (<2 ml) microwell-based experimental platform for the parallel investigation of ProSavin(®) production using suspension-adapted PS46.2. This is combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 µg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

Decreasing the cutoff for elevated blood lead (EBL) can decrease the screening sensitivity for EBL.

Change in the definition of elevated blood lead (EBL) from greater than or equal to 10 µg/dL (cutoff A) to greater than or equal to 5 µg/dL (cutoff B) was recently endorsed in the United States. A potential effect of this change is to decrease the screening sensitivity for EBL detection. We demonstrate this effect by simulated sampling of an example patient distribution for lead. Using lead-dependent assay imprecision, simulated sampling of the patient distribution tracked individual misclassifications relative to the EBL cutoff. Decreasing the EBL cutoff from A to B reduced screening sensitivity for EBL detection in this population to less than 90%, a decrease of 4%. The result was due to the fact that, for B, a greater fraction of the EBL population was near the EBL cutoff and therefore subject to misclassification due to assay imprecision. The effect of the decreased EBL cutoff to reduce EBL screening sensitivity is likely to apply to EBL screening programs generally.

Receiver-operating characteristics of adjusted urine measurements of oxycodone plus metabolites to distinguish between three different rates of oxycodone administration.

OBJECTIVES: In a study by Couto et al. (Use of an algorithm applied to urine drug screening to assess adherence to an oxycontin regimen. J Opioid Manag 2009;5:359-64), adjusted urine measurements of oxycodone plus metabolites noroxycodone and oxymorphone were determined among volunteer subjects in three groups according to oxycodone administration rates (A: 80 mg/day; B: 160 mg/day; C: 240 mg/day). We performed receiver-operating characteristic (ROC) analyses of the distribution data from this study to determine the ability to correctly categorize individual measurements with respect to each group. DESIGN AND METHODS: For groups A-C, assumed reference ranges were defined as median-centered intervals encompassing a designated central percentage of the group's distribution. By varying assumed reference ranges across all possible reference ranges, ROC analyses of the ability of each group's reference ranges to appropriately include or exclude members of all groups were calculated. This generated six ROC curves (sensitivity vs. specificity): A vs. (B or C); B vs. (A or C); C vs. (A or B). RESULTS: Overlaps of distributions A, B, and C were large, such that none of the ROC curves exceeded areas-under-curves of 0.8. The greatest sensitivity-specificity combination had a sensitivity of 74% for C with specificity of 75% for A, for which oxycodone administration rates were different by a factor of 3. CONCLUSIONS: ROC analyses of data from a previous study demonstrated that, even under experimentally controlled conditions, adjusted urine drug measurements could not be used reliably to correctly categorize individual subjects' results according to their known oxycodone administration rates in the range of 80-240 mg/day. Misclassifications of results were 25% or greater.

Predicted effects of proposed tightened acceptance criterion for Pb proficiency testing (PT) on PT sample pass rates for Pb point-of-care testing.

BACKGROUND: There is current advocacy for change in Pb proficiency testing (PT) acceptance criterion from ± 4 µg/dl ([Pb] <40 µg/dl; criterion a) to ± 2 µg/dl ([Pb] <20 µg/dl, criterion b). We examined the effect of this proposed change on PT sample pass rates for point-of-care testing (POCT) as predicted by imprecision of POCT PT sample results. METHODS: Inter-site standard deviations (s) of POCT PT results were tabulated as a function of [Pb] and characterized as a linear function of [Pb] (r(2)>0.8). Given s, predicted minimum, random-error-only PT failure rates (Fp) as a function of [Pb] were computed as the fraction of a normal distribution of results ([Pb]± s) that would fall outside of boundaries of acceptance criterion a or b. RESULTS: For [Pb]=2-20 µg/dl, current observed PT sample failure rates using criterion a range from 3 to 6%, which are greater than the predicted minimum failure rates based on s alone (Fp(a)=0-6%). In contrast, predicted minimum failure rates based on s using criterion b are greatly increased (Fp(b)=5-35%). CONCLUSIONS: Given the degree of inter-site imprecision among POCT Pb PT results, adoption of criterion b for PT acceptance will dramatically increase Pb PT sample failure rates for POCT due to random-error alone.