Ligands and Matrices The commercial availability of a range of carbohydrate-based supports enabled the expansion of chromatographic techniques.
At this point, the science largely bifurcated into ligand discovery and matrix improvement. Axén's9 introduction of cyanogen bromide activation in 1967 allowed the development of affinity chromatography, the invention of
which was attributed to Cuatrecasas et al. (1968).10 Interactions between Protein A and immunoglobulins were under investigation by Sjöqvist's11 group at Uppsala University in Sweden in the mid-1960s but IgG purification using Protein A adsorbents generally is ascribed
to the Lund researchers Hjelm et al.12 and Kronvall et al.13 Uppsala, however, is intrinsically linked to bioseparations from the time of The Svedberg (Nobel Prize, 1926), through the
activities of the Institute of Biochemistry (The Biomedical Centre) and the research and product development at Pharmacia
Fine Chemicals, now part of GE Healthcare. The drawbacks of hydrophobic Amberlite IRC-5014 and Dowex resins for protein separations gave rise to the search in the 1950s for matrices that did not interfere with the
separation on derivatized gels. In 1947, Boscott15 had described the use of solvent-treated cellulose acetate as a "satisfactory stationary organic phase for chromatography,"
which Howard and Martin termed "reversed-phase partition chromatography" (RPC).16 RPC has a continuous polar stationary phase and requires organic solvents, whereas hydrophobic interaction chromatography
(HIC) has polar ligands substituted onto a neutral backbone and is run with an aqueous mobile phase. Although these related
technologies of RPC and HIC were born in the 1950s, they did not come to commercial use until considerably later. 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.
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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.
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