Richard Fike

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.

Recombinant Protein Production by CHO Cells Cultured in a Chemically Defined Medium

Serum-free culture of Chinese hamster ovary (CHO) cells has become increasingly common as a way of obtaining high levels of expression of recombinant proteins while simplifying recovery and downstream processing of the product. However, serum-free media may still contain one or more of a variety of animal-derived components including albumin, fetuin, various hormones and other proteins. We have demonstrated that it is possible to eliminate animal-derived proteins from a CHO medium formulation. Plasma protein fractions like albumin and fetuin may be replaced by plant-derived hydrolysates, resulting in medium that is protein-free but still undefined (CHO III PFM). CD CHO Medium is a chemically defined formulation which contains no protein or hydrolysates of either plant or animal origin. Peak cell densities and recombinant protein expression in CD CHO cultures compared favorably to expression in other media, although the maximal cell density and the highest levels of expression were observed at later time points. We were able to successfully supplement the culture with sodium butyrate to increase expression levels at the expense of peak cell density, so for recombinant cell lines showing an inverse relationship between growth and expression of recombinant product, strategies which limit the peak cell density may be useful for increasing expression.

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.

A Novel Application of Granulation Technology to Improve Physical Properties and Biological Performance of Powdered Serum-Free Culture Media

The Advanced Granulation Technology (AGT) process was evaluated using serum-free and protein-free formulations containing ingredients believed to be sensitive to the ball-milling process. The AGT manufacturing process was scalable, based upon physical parameters, biochemical homogeneity and biological performance criteria. Cell culture growth and biological production studies involving CHO, Vero, hybridoma and HEK 293 cells demonstrated that serum-free/protein-free media derived from the AGT process supported equivalent or superior performance to identical formulations produced by traditional processes. AGT meets pharmaceutical quality guidelines and can be applied to a broad range of nutrient formulations used in bioproduction applications.

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).

Improved bioreactor productivity and manufacturing efficiency using liquid medium concentrates

Synopsis Liquid media concentrates (LMC), designed for a broad range of classical and novel culture formulations, have exhibited superior performance in large-scale animal cell bioreactors relative to conventional media delivery options. Various options for concentrate reconstitution reduce the complexity and cost of high volume media preparation. Collaborative field studies conducted over the past two years confirm that use of LMC can improve bioreactor productivity and manufacturing efficiency in a broad range of animal cell culture environments.

Technical Approaches to Minimize Regulatory Hurdles to Fed-Batch and Continuous Medium Supplementation

Mammalian cell culture biological production applications have traditionally implemented batch production techniques to minimize real or perceived obstacles to product approval by regulatory agencies. Analysis of the bioreactor environment during the course of a batch fermentation run demonstrates significant fluctuations in levels of critically-limiting nutrients, accumulation of catabolic products and increased medium osmolality. These factors can be demonstrated to impact specific productivity and to result in product microheterogeneity. Extended bioreactor campaigns may exhibit superior productivity and longevity by controlled nutrient delivery via fed batch or continuous perfusion of concentrated supplements. Regulations defining criteria for “well-characterized products” and international efforts to harmonize standards may minimize barriers to extended production campaigns and process modifications. This paper examines the evolution of these regulatory guidelines and their application to process changes which permit productivity enhancement through supplementation with salt-free nutrient concentrates.