Development of cell and tissue therapies presents bottlenecks in manufacturing process development and scale-up as commercial and academic groups move from small-scale research and development (R&D) to more complex logistics. Often, the simplicity of maintaining cell yield, viability, and function in a laboratory setting cannot be replicated when source tissue and final therapeutic products are subjected to the extended distances and times of actual clinical delivery.

These bottleneck issues have a number of causes. One specific and common cause is suboptimal biopreservation workflow processes and practices. At BioLife Solutions, we often address this particular issue in consultations with current and prospective customers, where we've heard the title of this article from a number of people. The following case study — a hypothetical amalgam drawn from our experiences — highlights various biopreservation workflow process optimization steps that can extend stability. This may improve postpreservation viability and functional yield of cellular source materials and final cell-based products.

PRODUCT FOCUS: ALL BIOLOGICS

PROCESS FOCUS: MANUFACTURING

WHO SHOULD READ: QUALITY, PROCESS DEVELOPMENT, ANALYTICAL, AND REGULATORY AFFAIRS

KEYWORDS: CRITICAL QUALITY ATTRIBUTES (CQAs), CRITICAL PROCESS PARAMETERS (CPPs), STATISTICAL ANALYSIS, VARIATION, PROCESS OPTIMIZATION, MODELING, BIOPRESERVATION

LEVEL: INTERMEDIATE

Case Study

HairCell Technologies has built on the well recognized understanding that stem cells derived from bone marrow can be used to treat a number of disorders, and the company's scientists used such cells to develop a proprietary technology that will improve the treatment of hair follicle disease. But during the development of HCT's technology beyond the R&D stage, several stability-related issues arose that threatened to doom this promising technology as not commercially feasible.

HCT's phase 1 clinical trial was being conducted in Asia, but the company's cell processing facility is located in the United States. Development scientists did not realize that such large distances involved in transport of both the source bone marrow and the finished cell therapy product would cause significant yield loss in the materials. They had assumed that an isotonic saline solution would be the natural transport medium for maintenance of cells during ambient shipment. But in fact, there was ~50% loss of cells in the source bone marrow during the first transport stage alone, and isolated stem cells exhibited a significant lag in regrowth.

HCT also observed significant biopreservation issues while manufacturing the two-stage final product. The company banked initially harvested cells as a cryopreserved product to extend its time management profile and allow for patient scheduling convenience. Upon scheduling the final (transplantation) procedure, that frozen product is then thawed at the US facility, cultured for 48 hours in a proprietary activation media, and shipped to Asia for patient application. This not only allows for time management of the final procedure, but it also allows for centralized processing independent of patient location. However, HCT found that cryopreservation of its cell therapy product in the standard cocktail of 10% dimethyl sulfoxide (DMSO) in saline resulted in ~55% cell loss over the 48-hour postthaw activation period. Furthermore, subsequent transport of activated cells in saline resulted in an additional 50% cell loss, with ultimately little evidence of functional utility in patients. HCT's executives put a hold on the clinical trial, and the company's primary investor threatened to withdraw continued funding.

HCT faced a bottleneck regarding its source material and final product stability constraints that many involved in cell therapy development might relate to — although perhaps not to the severity of this example. Common nonoptimized process development systems in regenerative medicine tolerate appreciable levels of cell damage/loss, and companies adjust culture times and applications around those limitations. Consider that use of many cryopreserved products often compensates for significant postthaw cell loss by merely increasing the cell concentration or volume of the clinical dose. (But who wants more dead cells injected into his or her body?).

The Challenges: HCT encountered the biopreservation limitations of cellular materials removed from normothermic conditions that provide resources for normal cell function. Biologics begin to degrade once they are removed from a living source organism (cell culture systems and conditions were developed to substitute for natural growth conditions). The sensitivity of cells to the stresses of being removed from their normal conditions is as varied as the different types and functions of the cells within an organism. Consider that kidneys can be preserved for days in a transplant process, but hearts can be preserved only for hours in clinical practice. To slow the degradation of cells and tissues, standard methods for transport or banking involve low temperatures in either hypothermic (2–8 °C) or cryopreservation (most often below –150 °C) ranges. But assuming that the same principles of normothermic biology apply to low-temperature cell models is a common misunderstanding and a critical pitfall encountered in nonoptimized process development.