Cell Culture

Growing and culturing of cells are the most fundamental steps in clone screening. The health of the cells is critical to optimizing the production of the protein of interest. Both adherent and suspension cells must be grown in an aseptic environment to ensure no contaminant is introduced that could render the batch unusable or reduce the integrity of subsequent analysis. In addition to an aseptic environment, growing media and nutrients supplemented to the cells must be sterile or else risk contamination.

Pall Laboratory offers a complete range of 0.2 µm sterile grade filtration solutions for cell culture applications from small feed volumes of a few milliliters, to over 100 liters. Our proprietary dual layer membranes enable fast and efficient filtration of nutrient rich media, feed, and supplements. Pall filtering media are low protein binding, ensuring valuable nutrients are not lost during sterile filtration. Sterile syringe filters allow small samples to be taken for assessment when monitoring culture growth without risk of contamination.

Mycoplasmas are a common contaminant of cell cultures but are not readily discernible by routine laboratory methods. Using our 0.1 µm Supor® mycoplasma reduction membrane is the best assurance to limit potential mycoplasma from being introduced into cell cultures by serum or serum-containing media. Pall Laboratory has filter products suitable to most workflow needs throughout the cell passaging and bioreactor culture process.

Cell
Harvesting
& Clarification

During harvesting of cell cultures to extract extracellular proteins, clarification is required to remove cells, cell debris, and large protein aggregates from the culture media prior to sterile filtration and purification. If not removed, the phospholipid bilayer of cell membranes can cause issues during downstream purification steps, decreasing the quality and quantity of protein products. Separation through centrifugation can be cumbersome for large volumes. Additionally, poorly clarified solutions can cause clogging during sterile filtration, slowing workflow. The use of small-pore filters during clarification and prior to sterilization can lead to cost reduction and improved product recovery. Establishing the strongest performing harvest process with the best fit technology for a specific cell culture early in monoclonal antibody (mAb) process development is essential. Scalability of the chosen technology is key for a seamless transition from small volumes to liters with minimal changes in process operation to ensure expected reproducible outcomes.

Pall Laboratory offers a range of depth filtration capsules with stacked and integrated high-performance depth media, designed to efficiently remove particulate contamination and clarify solutions prior to downstream processing. Depth filtration efficiently processes concentrated solutions with high cell counts such as cell cultures and fermentation broths. A third dimension, thickness, is added to the filter structure effectively trapping particulate matter, both at the surface and throughout the depth of the entire filter media. The correct choice of filter type allows for removal of cells of differing sizes (e.g. bacteria, yeast, and mammalian cells), and clarification of cell debris or cell lysate. Our Supracap™ Depth Filter Capsules are optimized for biological and pharmaceutical requirements and are scalable for GMP production requirements and size. Pall has designed a system for clarifying cell culture media that is well suited for monoclonal antibody (mAb) production that places a PDP8 Supracap filter in series with a PDE2 Supracap filter, the same configuration as Pall Stax™ mAx Clarification Platform that can handle hundreds of liters of media at a time.

Sterile 0.2 µm Filtration

Due to the nature of biopharmaceuticals, terminal sterilization is not possible, efficient sterile filtration through a 0.2 µm filter is essential to ensure biological contamination is removed prior to downstream processing. Sterile filtration will reduce the risk of the subsequent growth of microorganisms in the solution increasing sample stability before purification of the target protein

Choosing the right filtration system can significantly improve workflow efficiency and reduce cost. Selecting a system where optimized filtration parameters can be scaled up to commercial requirements, reduces the risks of impacts on Critical Quality Attributes of the product due to changes in established processes at a later stage.

At Pall Laboratory we can help you to select the optimal filtration system for your application from our range of high-performance dual-layer sterile filters. Our filters can reduce filtration time, increase throughput, reduce the need for multiple filter changes, and save in overall filtration costs.

Our AcroPak capsule products range in size from 20 cm2 to 1,500 cm2 effective filtration area (EFA). Three membranes choices are available in configurations that scale-up to products for pilot and full-scale production.

AcroPak Products with Supor® PES Membrane quickly process difficult-to-filter solutions such as viscous or particulate-laden solutions. Supor membrane offers high flow rates and consistently higher total solution throughputs due to higher porosity than most other membranes. It is ideal in situations where rapid filtration or short processing times are essential or where low protein binding is required.

Purification

The goal of the purification step in clone optimization is to enhance recovery of the target biomolecule for detailed functional analysis. Contaminants such as whole cell proteins, DNA, or unwanted mAb variants are removed through a series of chromatographic steps. The process can be quickened, and cost reduced, by concentrating the solution prior to purification.

To ensure the target proteins are in the desired solution for purification or analysis, diafiltration, a process of simultaneous buffer exchange and concentration can be performed before or after the chromatography step.

Ultrafiltration via Pall Laboratory Tangential Flow Filtration (TFF) systems provide efficient processing, concentration and diafiltration (desalting or buffer exchange) on the same system with minimal user intervention. Optimized flow path design coupled with low protein-binding membranes minimize non-specific binding for high product recovery. Our scalable range of TFF capsules delivers predictable performance saving time when scaling up the process. Concentration and buffer exchange can be done simultaneously in a process called ‘continuous-diafiltration’ which can save hours of processing time.

Functional Analysis

In addition to identifying clone candidates that stably produce the biotherapeutic protein, the purity, titer and biological activity of the biomolecule is analyzed as part of an iterative process to optimize production. Functional analysis includes protein transcription, expression, structure and cellular interaction studies through techniques such as polyacrylamide gel electrophoresis (SDS-PAGE), Liquid Chromatography–Mass Spectrometry (LC-MS) and High-Performance Liquid Chromatography (HPLC).

Pall Laboratory offers innovative nucleic acid binding centrifugal devices which simplify protein sample preparation, gDNA and mRNA purification prior to LC-MS or HPLC analysis. The Nanosep® Centrifugal Filter incorporates a high-binding innovative double layer glass fiber membrane. High-quality and quantity yields with even recovery of fragments as small as 50 bp up to 10, 000 bp, support multiple downstream applications, while saving time and resource by removing the need to process additional samples.

High-sensitivity, low background noise membranes designed for protein analysis applications, such as western blotting, provide clarity of results. Immobilized proteins to be used directly for sequencing increasing flexibility in your workflow. Products are optimized to reduce extractables and pre-sterilization by gamma irradiation eliminates potential cytotoxic residues.