Biological Matrix Supply Chain Shortages: More Matrices Are Now Rare-the Case for Surrogate Matrices.

The COVID-19 pandemic has strained the biological matrix supply chain. An upsurge in demand driven by numerous COVID-19 therapeutic and vaccine development programs to combat the pandemic, along with logistical challenges sourcing and transporting matrix, has led to increased lead times for multiple matrices. Biological matrix shortages can potentially cause significant delays in drug development programs across the pharmaceutical and biotechnology industry. Given the current circumstances, discussion is warranted around what will likely be increased use of surrogate matrices in support of pharmacokinetic (PK), immunogenicity, and biomarker assays for regulatory filings. Regulatory authorities permit the use of surrogate matrix in bioanalytical methods in instances where matrix is rare or difficult to obtain, as long as the surrogate is appropriately selected and scientifically justified. Herein, the scientific justification and possible regulatory implications of employing surrogate matrix in PK, immunogenicity, and biomarker assays are discussed. In addition, the unique challenges that cell and gene therapy (C>) and other innovative therapeutic modalities place on matrix supply chains are outlined. Matrix suppliers and contract research organizations (CROs) are actively implementing mitigation strategies to alleviate the current strain on the matrix supply chain and better prepare the industry for any future unexpected strains. To maintain ethical standards, these mitigation strategies include projecting matrix needs with suppliers at least 6 months in advance and writing or updating study protocols to allow for additional matrix draws from study subjects and/or re-purposing of subject matrix from one drug development program to another.

Quartz crystal microbalance as an assay to detect anti-drug antibodies for the immunogenicity assessment of therapeutic biologics.

Because of their biological origins, therapeutic biologics can trigger an unwanted deleterious immune response with some patients. The immunogenicity of therapeutic biologics can affect drug efficacy and patient safety by the production of circulating anti-drug antibodies (ADA). In this study, quartz crystal microbalance (QCM) was developed as an assay to detect ADA. Etanercept (Enbrel®) was covalently grafted to dextran-modified QCM surfaces. Rabbits were immunized with etanercept to generate ADA. Results showed the QCM assay could detect purified ADA from rabbits at concentrations as low as 50 ng/mL, within the sensitivity range of ELISA. The QCM assay could also assess the ADA isotype. It was shown that the ADA were composed of the IgG isotype, but not IgM, as expected. Furthermore, it was shown that QCM surfaces that had been used to detect ADA could be regenerated in glycine-HCl solution and reused. The QCM assay was also demonstrated to detect ADA in crude serum samples. Serum was collected from the rabbits and analyzed before and after etanercept immunization. ADA were clearly detected in serum from rabbits after immunization, but not in serum before immunization. Serum from patients administered with etanercept for rheumatoid arthritis (RA) treatment was also analyzed and compared to serum from healthy donors. Sera from 10 RA patients were analyzed. Results showed one of the RA patient serum samples may have ADA present. In conclusion, QCM appears to be a viable assay to detect ADA for the immunogenicity assessment of therapeutic biologics.

Real-time label-free detection and kinetic analysis of Etanercept-Protein A interactions using quartz crystal microbalance.

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel®) into the QCM chambers. The kinetics of Enbrel® - Protein A interactions was modeled using the Langmuir binding model and Enbrel® concentrations between 0.75-300ngmL-1. The resulting equilibrium dissociation and association constants (KD and KA) were 5.06×10-8M and 1.98×107M-1, respectively. The association and dissociation rate constants (kon and koff) decreased substantially as Enbrel® concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in kon and koff was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel® concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in kon and koff for increasing Enbrel® concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.

INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor.

Understanding ß cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic ß cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR + GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion.

Solution composition impacts fibronectin immobilization on carboxymethyl-dextran surfaces and INS-1 insulin secretion.

It is shown that solution composition during immobilization plays a critical role in the properties of fibronectin (FN) surfaces and their bioactivity towards insulinoma (INS-1) cell function. X-ray photoelectron spectroscopy revealed FN grafting onto low-fouling carboxymethyl-dextran (CMD) surfaces was successful with solutions composed of 10 µM CaCl(2), 10 µM MgCl(2), 10 µM MnCl(2), and 10 µM and 1mM NaCl, but unsuccessful with those made of 150 mM NaCl or 1× PBS. Circular dichroism and photon correlation spectroscopy revealed that regardless of solution composition, no measurable differences in free FN conformation prevail. AFM imaging of FN-CMD revealed, while there are no quantitative differences in surface roughness, there are some subtle qualitative differences in topography. FN surface immobilization scheme does not influence INS-1 cell growth after 3 and 7 days regardless of the underlying substrate or solution composition. INS-1 cell insulin secretion in response to glucose is affected by the substrate and solution composition during FN immobilization.

In vitro morphogenesis of PANC-1 cells into islet-like aggregates using RGD-covered dextran derivative surfaces.

Substrates bearing low-fouling carboxymethyl dextran (CMD) upon which RGD is covalently grafted were validated to study PANC-1 cell differentiation in serum-free medium. When exposed to RGD-CMD, cells lightly adhered to the surface and formed islet-like aggregates (ILAs) in contact with the surface. PANC-1 were non-adherent on RGE-CMD, CMD, and tissue culture polystyrene surfaces and aggregated in suspension forming ILAs. RGD-CMD resulted in smaller ILAs. Ki67 and CK-19 expression decreased after 3, 7, and 14 days. E-cadherin expression did not change from 3 to 7 days, but decreased after 14 days. For each marker, there was no difference in the expression between the surfaces. After 14 days, ILAs weakly expressed insulin and glucagon and the expression level was independent of the surfaces. No convincing level of insulin or glucagon was detected by immunostaining at 3 and 7 days. A higher level of insulin release was detected in the media after 14 days for cells grown on RGD-CMD. After 14 days, there were three-fold more cells on RGD-CMD surfaces compared to cell numbers found in other culture conditions. α(5) integrin expression was rampant throughout ILAs for all four surfaces. α(V)ß(3) integrin expression was only noticeable for ILAs cultured on RGD-CMD surfaces.

Culturing INS-1 cells on CDPGYIGSR-, RGD- and fibronectin surfaces improves insulin secretion and cell proliferation.

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg-Gly-Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins αvß3 and α5 were similar between the conditions, except for α5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1.