An Intercompany Perspective on Practical Experiences of Predicting, Optimizing and Analyzing High Concentration Biologic Therapeutic Formulations.

Developing high-dose biologic drugs for subcutaneous injection often requires high-concentration formulations and optimizing viscosity, solubility, and stability while overcoming analytical, manufacturing, and administration challenges. To understand industry approaches for developing high-concentration formulations, the Formulation Workstream of the BioPhorum Development Group, an industry-wide consortium, conducted an inter-company collaborative exercise which included several surveys. This collaboration provided an industry perspective, experience, and insight into the practicalities for developing high-concentration biologics. To understand solubility and viscosity, companies desire predictive tools, but experience indicates that these are not reliable and experimental strategies are best. Similarly, most companies prefer accelerated and stress stability studies to in-silico or biophysical-based prediction methods to assess aggregation. In addition, optimization of primary container-closure and devices are pursued to mitigate challenges associated with high viscosity of the formulation. Formulation strategies including excipient selection and application of studies at low concentration to high-concentration formulations are reported. Finally, analytical approaches to high concentration formulations are presented. The survey suggests that although prediction of viscosity, solubility, and long-term stability is desirable, the outcome can be inconsistent and molecule dependent. Significant experimental studies are required to confirm robust product definition as modeling at low protein concentrations will not necessarily extrapolate to high concentration formulations.

An Intercompany Perspective on Compatibility and In-use Stability Studies to Enable Administration Of Biopharmaceutical Drug Products.

In-use stability and compatibility studies are often used in biotherapeutic development to assess stability and compatibility of biologic drugs with diluents and/or administration components at relevant conditions for the target route of administration (commonly intravenous, subcutaneous or intramuscular), to assure that patient safety and product efficacy are maintained during clinical use. To gain an understanding of current industry approaches for in-use stability and compatibility studies, the Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, conducted an inter-company collaboration exercise, which included five bench-marking surveys around in-use stability and compatibility studies of biologic drugs. The results of this industry collaboration provide insights into the practicalities of these studies and how they are being used to support administration of biologics from early clinical programs to marketed products. The surveys queried topics including regulatory strategies and feedback; clinical in-use formulation, patient and site considerations; clinical blinding, masking and placebo approaches; study setup, execution and reporting; and clinical in-use stability and compatibility testing to provide a comprehensive picture of the range of common industry practices. This paper discusses the survey results and presents various approaches which can be used to guide the strategy and design of an in-use stability and compatibility program based on clinical and biomolecule needs.

Clusterin glycopeptide variant characterization reveals significant site-specific glycan changes in the plasma of clear cell renal cell carcinoma.

Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients. Here, we characterized the occupancy and the degree of heterogeneity of individual N-glycosylation sites of clusterin in the plasma of patients diagnosed with localized ccRCC, before and after curative nephrectomy (n = 40). To this end, we used tandem mass spectrometry of immunoaffinity-enriched plasma samples to analyze the individual glycosylation sites in clusterin. We determined the levels of targeted clusterin glycoforms containing either a biantennary digalactosylated disialylated (A2G2S2) glycan or a core fucosylated biantennary digalactosylated disialylated (FA2G2S2) glycan at N-glycosite N374. We showed that the presence of these two clusterin glycoforms differed significantly in the plasma of patients prior to and after curative nephrectomy for localized ccRCC. Removal of ccRCC led to a significant increase in the levels of both FA2G2S2 and A2G2S2 glycans in plasma clusterin. These changes were further confirmed by lectin blotting of plasma clusterin. It is envisioned that these identified glycan alterations may provide an additional level of therapeutic or biomarker sensitivity than levels currently achievable by monitoring expression differences alone.

Development of an improved fractionation of the human plasma proteome by a combination of abundant proteins depletion and multi-lectin affinity chromatography.

AIM: Current analytical tools lack the required capacity to reduce the complexity of the plasma proteome and identify low-level proteins of clinical interest. Hence, the need to develop a fractionation approach to provide adequate throughput for a clinical study and minimize the loss and improve the detection of low abundance proteins. MATERIALS & METHODS: We present the development of an analytical platform that combines the depletion of 12 high abundance proteins and multi-lectin affinity chromatography (12P-M-LAC) fractionation. RESULTS & CONCLUSION: We validated the highly specific, stable and robust 12P-M-LAC platform using human plasma. An improved enrichment of low abundance proteins and glycoproteins with minimum sample loss was achieved demonstrating the suitability of this platform in future biomarker discovery studies.

Comparative studies of the proteome, glycoproteome, and N-glycome of clear cell renal cell carcinoma plasma before and after curative nephrectomy.

Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor disease recurrence. Here, we report on the utilization of a multi-dimensional fractionation approach (12P-M-LAC) and LC-MS/MS to comprehensively investigate clear cell renal cell carcinoma plasma collected before (disease) and after (non-disease) curative nephrectomy (n = 40). Proteins detected in the subproteomes were investigated via label-free quantification. Protein abundance analysis revealed a number of low-level proteins with significant differential expression levels in disease samples, including HSPG2, CD146, ECM1, SELL, SYNE1, and VCAM1. Importantly, we observed a strong correlation between differentially expressed proteins and clinical status of the patient. Investigation of the glycoproteome returned 13 candidate glycoproteins with significant differential M-LAC column binding. Qualitative analysis indicated that 62% of selected candidate glycoproteins showed higher levels (upregulation) in M-LAC bound fraction of disease samples. This observation was further confirmed by released N-glycans data in which 53% of identified N-glycans were present at different levels in plasma in the disease vs non-disease samples. This striking result demonstrates the potential for significant protein glycosylation alterations in clear cell renal cell carcinoma cancer plasma. With future validation in a larger cohort, information derived from this study may lead to the development of clear cell renal cell carcinoma candidate biomarkers.