Neutral resins for HIC were developed as a result of the work of Porath17 and Hjertén,18 who also introduced the accepted name of the technique, and products became available as late as 1977. Also, in 1972–73,
hydrocarbon-coated Sepharose derivatives were developed at the Weizman Institute.19 Reverse-phase separations took a development path through high-performance liquid chromatography (HPLC), driven by Horvath's
work starting in 1966 and his invention of the HPLC instrument.20 Largely due to the work of Kirkland21 at Dupont, bonded-phase silica became the matrix of choice for RP–HPLC; new stationary phases were developed for biomedical
applications in the 1980s. HPLC is now one of the most accepted techniques.
Hancock notes that as an analytical method,"RP–HPLC played a key role at Genentech in the development of rhHGH as a pharmaceutical,"22 and RP–HPLC has continued to play that key role in product development and control in biopharmaceutical laboratories around
the world. For protein separation at an industrial scale, however, HPLC is more limited in its applicability because of the
need for organic solvents and because of the pressure demands in an industry that otherwise operates below 3 bar. The notable
exception is Eli Lilly's use of the technology for purifying biosynthetic human insulin (as it was called at the time).23
Scale-Up
Figure 1. A 2,500-L "Sephamatic Gel Filter" packed with Sephadex G-25 used for the production of a desalted whey protein
concentrate (free from lactose and salts) in 1968. (COURTESY OF GE HEALTHCARE)
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Low-pressure process chromatography could not have developed without immense efforts to resolve scale-up issues in both column
design and matrix stability. Early work in scale-up was thus restricted to the use of rigid gels such as Sephadex G-25 in
stainless steel columns or "Gel Filters," which were developed and introduced in 1968 by Pharmacia Fine Chemicals (Figure
1). Efforts were being made to overcome the pressure-flow restrictions of soft gels, and work by Janson24 led to the commercialization of the "Stack" or sectional column. The column dimensions were 16 cm bed height by 37 cm diameter,
only because this was the largest polypropylene mold size that could be made at the time. These early columns had fixed bed
heights and the gel filters could be pump packed, predating today's packing methods by several decades.
Improving Media
Figure 2. The "Stack" column used for insulin purification in the 1970s. Single sections of this column became standard in
the pilot scale use of ion exchangers for, for example, plasma protein purification. The 16-cm bed height was a driving force
in the move to short bed columns and the scale up to a 30 cm x 150 cm bed column as standard in the 1970s. (Courtesy of GE
Healthcare, originally from CSL Ltd, Melbourne, Australia.)
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Since the late 1960s, manufacturers of chromatographic resins have developed increasingly robust media for process scale chromatography.
They continue to search for improvements in stationary phases to keep pace with the increasing demands of the biotechnology
industry for improved product throughput. The development battleground was and still is overcoming mass transfer limitations
due to diffusion, in turn limited by residence time, bead porosity, bead size, and matrix morphology in the case of continuous
stationary phases.
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