Strategies for Setting Patient-Centric Commercial Specifications for Biotherapeutic Products.

Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance. For other product quality attributes (PQAs), product knowledge and the understanding of attribute criticality built throughout product development should drive specification setting. The foundation of PQA knowledge is an understanding of potential patient impact through an assessment of potency, PK, immunogenicity and safety. In addition to PQA knowledge, the ability of the manufacturing process to consistently meet specifications, typically assessed through statistical analyses, is an important consideration in the specification-setting process. Setting acceptance criteria that are unnecessarily narrow can impact the ability to supply product or prohibit consideration of future convenient dosage forms. Patient-centric specifications enable appropriate control over higher risk PQAs to ensure product quality for the patient, and flexibility for lower risk PQAs for a sustainable supply chain. This paper captures common strategic approaches for setting specifications for standard biotherapeutic products such as monoclonal antibodies and includes considerations for ensuring specifications are patient centric.

Native size-exclusion chromatography-mass spectrometry: suitability for antibody-drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels.

Native size-exclusion chromatography-mass spectrometry (nSEC-MS) is an analytical methodology that is appropriate for accurately quantitating the drug-to-antibody ratio (DAR) on a wide variety of interchain cysteine-linked antibody-drug conjugates (ADCs), irrespective of chemotype. In the current preclinical environment, novel ADCs conjugated with unique drug-linkers need to progress toward the clinic as quickly as possible. Platform analytical approaches can reduce time-to-clinic because key process development and optimization activities can be decoupled from the development of bespoke, molecule-specific analytical methods. In this work, we assessed the potential of nSEC-MS as a platformable, quantitative DAR method. The nSEC-MS method was evaluated according to performance characteristics and parameters described in the ICH guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). In order to comprehensively assess the accuracy and bias of nSEC-MS DAR quantitation, ADCs were generated using three different drug-linker chemotypes with DARs ranging from 2 to 8. These molecules were tested by hydrophobic interaction chromatography (HIC) and nSEC-MS, and DARs obtained from both methods were compared to assess the degree to which nSEC-MS quantitation aligned with the HIC release assay. Our results indicated that there is no bias introduced by nSEC-MS quantitation of DAR and that SEC-MS data can be bridged to HIC data without the need for a correction factor or offset. nSEC-MS was also found to be suitable for unbiased DAR quantitation in the other ADC chemotypes that were evaluated. Based on the totality of our work, we conclude that, used as intended, nSEC-MS is well suited for quantitating DAR on a variety of interchain cysteine-linked ADCs in an accurate, unbiased manner.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity.

Routine CHO cell line development practices involve a lengthy process of iteratively screening clonally derived cell lines to identify a single line suitable for IND filing and clinical manufacture. Paramount in this process is development of a stable production cell line having consistent growth, productivity and product quality for the entire generational length of the manufacturing process. Scale-down stability models used to screen clones for consistency are time consuming and often a rate-limiting step in clone selection. To investigate CHEF1 production stability in CHO cells we analyzed genotypic and phenotypic attributes of monoclonal primary clones and their respective subclones over time in standard antibody production models. The main finding of this work indicates that monoclonal cell lines derived from single cell progenitors grow into populations of cells with varied phenotypic heterogeneity, as revealed in their subclones, from either stable or unstable cell lines. Investigation of the subclones demonstrates that clonally derived cell lines grow out into populations with variable phenotypes and genotypes, even if the primary clone shows consistency in both over many generations in a stability study. Phenotypic and genotypic heterogeneity mostly did not correlate, but growth and productivity appear driven in part by cytosine methylation heterogeneity in both primary and secondary clones. This work presents evidence that epigenetic analysis may be useful for early detection of stability traits, but emphasizes the continued importance of rigorous cell line stability screening to identify primary clones that have consistent phenotypic characteristics, especially growth and productivity, throughout the in vitro lifecycle of the cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:635-649, 2018.

HIV-associated nephropathy and end-stage renal disease in children in the United States.

Single-center studies have reported that HIV-associated nephropathy (HIVAN) can occur in children and may have a clinical course and prognosis similar to that of adults. However, the prevalence and survival has not been reported for a national sample of children with HIVAN and end-stage renal disease (ESRD) on dialysis in the United States. We utilized the United States Renal Data System (USRDS) database to determine the prevalence, demographic information, and survival of children with HIVAN and ESRD in the United States. The Kaplan-Meier method was used to estimate survival of children with HIVAN and the log-rank test was used to compare their survival with children with focal segmental glomerulosclerosis (FSGS) and adults with HIVAN. Cox regression analysis was used to model adjusted hazard ratios (AHR) with HIVAN as a cause of ESRD and its impact on mortality during the study period, adjusted for potential confounders. Of the 7,732 patients identified with HIVAN, only 60 were younger than 21 years and were classified as children; 50% were males and the majority (88.3%) was black. The cumulative percentage survival of children with HIVAN at 12, 24, and 36 months was better than adults with HIVAN (76%, 62%, and 54% vs. 60%, 43%, and 34%). Survival of children with HIVAN who started dialysis after 1996 was significantly better than those who started dialysis in or before 1996 (log rank P value <0.043). However, the major factor associated with better survival on Cox proportional hazard analysis was female gender (male vs. female AHR 2.85, 95% confidence interval 1.04-6.73). We conclude that only a small number of children with HIVAN and ESRD have received dialysis in the United States. The prognosis of these children is better than that of adults with HIVAN and among children with HIVAN females have better survival than males.