David W. Jayme

Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture

Technical limitations and evolution of therapeuticapplications for cell culture-derived products haveaccelerated elimination of animal-derived constituentsto minimize inadvertent introduction of adventitiousviral or prion agents. Practical considerationsdemand adequate emphasis both on design of theserum-free/protein-free culture environment and onnutrient media manufacturing process controls. Protein components may be acceptable, given adequateattention to synthetic process, sourcing (e.g.,geographic location and endemicity, species andtissue/organ) and validated treatment method. Variousoptions exist for re-engineering of traditionalserum-free formulations (containing insulin,transferrin and other protein factors) withnon-protein substitutes. Caution must also beexercised with sourcing of non-protein additives,particularly amino acids and lipids, to avoidintroducing adventitious contaminants. Simpleguidelines facilitate adaptation, cryopreservation andrecovery of many cell types within a protein-freeculture environment. Scrupulous maintenance offacility and equipment and monitoring of processwater, air handling systems and technical personnelare required to ensure that approved raw materials arecorrectly formulated and dispensed. Validatedsanitization processes provide additional assuranceagainst cross-contamination from previous batches ina multi-use facility.

Growth of NS0 Cells in Protein-Free, Chemically Defined Medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein‐free media. Compared with serum‐supplemented media, use of protein‐free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum‐supplemented medium or a serum‐free medium supplemented with an animal‐derived lipoprotein. CD Hybridoma Medium (a protein‐free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX‐CYTE VLE. None of the tested supplements supported long‐term growth of NS0 cells in CD Hybridoma Medium. Sustained long‐term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal‐ or plant‐derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin‐lipid complex reached peak cell densities that were more than double those observed in serum‐supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin‐based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein‐free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal‐derived components if the supplement is made with plant‐derived or synthetic lipids.

Nutrient optimization for high density biological production applications

ConclusionAt the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old.This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs.It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.

Basal medium development for serum-free culture: a historical perspective

The evolution of basal synthetic formulations to support mammalian cell culture applications has been facilitated by the contributions of many investigators. Definition of minimally-required nutrient categories by Harry Eagle in the 1950s spawned an iterative process of continuous modification and refinement of the exogenous environment to cultivate new cell types and to support emerging applications of cultured mammalian cells. Key historical elements are traced, leading to the development of high potency, basal nutrient formulations capable of sustaining serum-free proliferation and biological production. Emerging techniques for alimentation of fed batch and continuous perfusion bioreactors, using partial nutrient concentrates deduced from spent medium analysis, can enhance medium utilization and bioreactor productivity.

Use of Liquid Medium Concentrates to Enhance Biological Productivity.

Liquid medium concentrates (LMC) were initially developed in response to industrial customer demand for improved efficiency and productivity of mammalian cell bioreactors. The resultant technology, which is undergoing its second generation of improvement, exploits biochemical properties intrinsic to the constituent nutrients to improve solubility, stability, and biological performance. Customer-perceived benefits, relative to liquid media produced by conventional technologies, include both enhanced biological productivity and improved manufacturing efficiency. The key concept which drives superior performance is that in concentrate technology all nutrient components of a complex biochemical formulation are fully pre-solubilized as a minimal number (generally three) of 50X LMC sub-groupings precedent to complete admixture. Under conventional procedures, critical nutrient potencies may be reduced relative to theoretical values due to poor solubilization and partial removal by precipitation and filtration. Use of LMC intermediates for the formulation of liquid media produces superior initial correlation with theoretical nutrient potency and may result in superior performance stability. Delivery of the full complement of nutrients to the biomass may be limiting to culture survival, attainment of maximal cell density, optimization of the specific cellular productivity, and prolongation of the bioreactor production cycle. The advantages of LMC become increasingly apparent with serum reduction or elimination, augmented cell densities, enhanced medium residence time and increasing development of balanced nutrient formulations. Complex nutrient media, formulated as LMC, are stable for up to one year in many cases, are compatible with various hulk containers, and exhibit superior biochemical potency and biological performance relative to conventionally-prepared culture media.

Continuous, high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.

Use of Medium Concentrates to Improve Bioreactor Productivity

Large-scale culture of recombinant mammalian or invertebrate cells requires significant volumes of nutrient medium. Batch preparation of liquid medium from powdered mixtures requires large stainless steel formulation tanks and is labor-intensive. Purchase of large volumes of 1X liquid medium, even in bulk containers, becomes economically unattractive for production of therapeutic quantities of biologicals. We have developed liquid medium concentrate technology which permits traditional synthetic media and serum-free formulations to be prepared in a minimal number of 50X component concentrates. The principles associated with concentrate technology may be extrapolated to a broad range of catalog and custom formulations. Technical advantages include: (1)improved stability of biochemical nutrients, including glutamine; (2)improved solubility of complex media constituents; and (3)enhanced cell growth rate, maximal density, and biological product yield relative to medium prepared directly from powder. Practical considerations include: (1)reduced refrigerated storage space; (2)increased nutrient medium shelf life; (3)improve efficiency of manufacturing facility space utilization; (4)reduced space, labor and equipment costs for media preparation; (5)decreased risk of aerosol-related health hazards and cross-contamination; (6)increased potential medium lot size; (7)reduced quality control testing costs; and (8)enhanced consistency in media formulation between remote manufacturing sites. Liquid medium concentrates appear compatible with large volume bulk containers. Standard mixing protocols permit batch reconstitution into single strength medium. Alternatively, a continuous flow mixing device permits on-line medium reconstitution for perfusion bioreactors.

Options to Eliminate Animal-Derived Components of Cell Culture Media

This paper has focused upon potential replacements for animal origin medium components which exhibit comparable biological performance in cell culture systems but eliminate concerns regarding introduction of adventitious contaminants. Nutrient media devoid of components derived from animals are commercially available. Similar strategies may be exploited to modify customized formulations to generate protein-free and even biochemically-defined second generation media to support cellular proliferation and biological production.

Cost-Saving Design and Operational Options for Large-Scale Production of Nutrient Medium and Buffers

Technical and economic advantages may derive from use of liquid concentrates to formulate nutrient medium and buffers. Batch reconstitution may be performed in formulation tanks, within customized bulk packaging, or directly within the bioreactor. Improvements in operating convenience, utilization efficiency of facility, equipment and technical personnel, and manufacturing cost were observed when concentrate reconstitution was performed using a mixing device for continuous delivery of diluted fluids directly into a bioreactor or into interim storage vessels. The preferred alternative may depend upon bioreactor culture conditions, volumetric consumption requirement, and buffer application and composition.

Productivity Enhancement Using Liquid Medium Concentrates

Qualitative and quantitative differences in nutrient utilization have been observed within high density mammalian cell culture bioreactors. Classical responses to depletion of critical nutrients are to increase the rate of total medium replenishment or to recirculate spent medium. An alternative is to maintain the exchange rate for basal medium components while adding rapidly-consumed nutrients as a supplemental concentrate. Composition and formulation of supplemental nutrient concentrates required quantitative analysis of spent biological fluids. Nutrient balance according to pseudo first order nutrient consumption kinetics, rather than post batch culture exhaustion profiles, yielded superior cell culture performance. Knowledge of nutrient biochemistry from partial supplements facilitated preparation of concentrated components to reconstitute complete media, including serum-free and protein-free formulations. Concentrated (50X) media exploited native properties to increase nutrient component solubility, sequester reactive materials, and permit biochemical co-stabilization. Liquid medium (1X) reconstituted from concentrated 50X sub-groups exhibited quality and performance advantages compared with identical formulations produced by classical options. This paper examines three key productivity indicators (cell yield, biological product yield, and overall media manufacturing cost).