James M. Kubiak

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.

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).

Continuous, Automated Reconstitution of Liquid Media Concentrates

Two years of customer experience with liquid media concentrates (LMC) have confirmed the superior consistency, quality and stability of IX liquid media produced from concentrated intermediates. However, facilitated processes and reduced cost for media manufacture have been the primary motivations for conversion by large volume industrial users from powdered medium to LMC. Recent technical advances have increased the breadth of formulations, enhanced lipid solubility and vitamin stability, increased fold concentration, and qualified alternative concentrate buffering approaches. Most large-scale users of LMC have reconstituted medium in formulation tanks similar to their current method for producing liquid medium from powder, experiencing a 50–75% reduction in overall time required for high volume medium manufacture. Some users have consistently reconstituted medium from LMC in closed vessels containing sterile diluent water. We now report development of an automated admix device for continuous apportionate reconstitution of LMC. The unit is designed for compatibility with a clean room environment, with size (6 ft2 of floor space) and weight (less than 400 lbs.) parameters to minimize facility costs. A prototype unit is capable of on-line preparation of liquid medium at continuously adjustable delivery rates up to 200 liters per hour with a mixing precision tolerance of less than ±1%. Its modular design permits regulated delivery of up to five LMC nutrient feeds. Second generation mixing devices based upon this prototype design are targeted for 1994 field evaluation to expand this capability to flow rates of 1500 liters per hour incorporating an unlimited number of independently-regulated LMC feed streams driven by a process logic controller and software interface to facilitate initial validation and batch documentation. Utilizing appropriate LMC intermediates, the mixing device can be applied to large volume preparation of nutrient medium for bioreactor feeding or of buffered salt solutions used for cell/tissue washing or chromatographic purification. Nutrient media reconstituted using this mixing device may be sterile-filtered and delivered either directly to a large surge tank or manifolded to multiple storage vessels. Technical advances in LMC sub-grouping and stabilization combined with the automated reconstitution capacity of the apportionate mixing device provides the potential for continuous preparation of a homogeneous 250,000 liter batch of liquid medium or buffered salt solution from bulk concentrates.

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.