Wh at considerations are necessary during the earlys t ages of liquid ch ro m at ograp hy process design toe n s u re successful scaleup?
Liquid chromatography is an essential purification step inachieving the high purities required for the products of larg e -scale biopharmaceutical production. However, at the produc-tion scale, this process has a different set of priorities andobjectives vs. its analytical and laboratory-scale counterparts.At the production scale, variations of the technique are increas-ingly being used to separate biomolecules in a complex mix-ture into individual components for analytical or quantitativepurposes, to isolate single biomolecules, and to eliminate con-taminants. Column loads are much higher and the loss of reso-lution due to overload may approach the limits of acceptableseparation quality. The information presented here addressesthe aspects of production-scale chromatography that may aidusers in process development, scaleup and final design of man-ufacturing-scale operations.The components of liquid chromatography are the same, nomatter what the type of separation or scale is involved. The sta-tionary phase (also called a sorbent or matrix) consists of smallparticles that are positively, negatively charged, or neutral,functional groups. The mobile phase (or eluant), a solution thatcarries the solute, is pumped into the stationary phase, whichhas been packed into the column, until complete binding equi-librium is reached.In some cases, a single mobile phase forms a mixture withthe feed components in which the constituents move throughthe column at various rates relative to the mobile phase,depending upon their degree of ionic interaction with thematrix. This mode of operation is referred to as isocratic/stepelution or gradient elution.In other cases, constituents may bind so tightly to the sor-bent that in order to elute them, the composition of the mobilephase must be changed by either increasing its ionic strength(
e . g .
, NaCl gradient) or changing its pH, either in discretesteps (stepwise elution) or in continuous mode (gradient elu-tion) until the components of interest begin to move throughthe column.
Production-scale chro m a t o g raphy considera t i o n s
Many commercial manufacturing processes opt for the reli-ability and low cost of ion-exchange chromatography (IEC) asa basic step. Hydrophobic-interaction and size-exclusion (gelfiltration) chromatographic stages (HIC, SEC) are often inte-grated to improve purification, but these techniques increasethe number of steps in the overall process, which is generallyundesirable. A product-specific binding technique, such asa ffinity chromatography (AC), captures very dilute compo-nents from a vast number of contaminants, concentrates theproduct, and does it all in fewer separation steps than eitherHIC or SEC. In certain cases, AC may be used alone to selec-tively remove the desired product molecules from the stream,while allowing the contaminants to flow through.Reversed-phase liquid chromatography (RPLC) uses verystrong hydrophobic interactions between the biologicalmolecule and the ligands on the chromatographic support toobtain separation. Elution requires highly concentrated org a n i csolvents, such as acetonitrile or 2-propanol, which may impairthe enzymatic and immunologic activity of the product protein,thereby lowering recovery rates. However, RPLC is sometimesthe only technique that can deliver a final separation for proteinenantiomers. In effect, the technique is somewhat limited to theseparation of peptides or for process control.The advantage of employing RPLC in large-scale opera-tions is that it employs a short column with a small diameter,yet it is equivalent to having a large number of equilibriumstages.This technique requires a higher capital investment thanmost other chromatography methods and is considered whenthe product is extremely valuable.
Production-scale purification
For larg e r-scale chromatography processes (pilot- or pro-duction-scale), purification becomes more challenging.Ye t ,the introduction of additional purification steps, such asmembrane separations, often translates into higher productlosses and lower overall product yield. Through the carefulselection of the medium and buffer for individual chromatog-raphy steps, and the employment of these steps in the propersequence, one can eliminate or reduce certain membrane sep-aration stages. Also, expanded-bed adsorption may be useddownstream to capture the product directly from the bioreac-tor broth, thereby limiting the number of separation stages(
e . g .
, microfiltration or centrifugation) and product concen-t r a t i o n / b u ffer exchange steps (
e . g .
, ultrafiltration) needed topurify the product.The one requirement that every chromatographic processmust meet, regardless of scale, is achieving a uniform flow dis-tribution across the cross-sectional area of the column, fromthe inlet to the outlet ports, with minimal mixing and dilutionin the flow-distribution chambers at either end of the column.Uniform flow distribution is easy to achieve with small-diameter laboratory columns, but as the diameter increases, sodoes the difficulty of maintaining uniform flo w. One possiblesolution is splitting the flow and distributing it through multi-ple ports on the column end-plates. But this may make it diff i-cult to clean and air lock one or more of the ports — a problemthat can be difficult to detect and correct. A second approach isto use a single port and distribute the flow through a system of radial ribs cut into the end-plates. For a larg e r-diameter col-umn, an anti-jetting device is used to reduce and disperse theentering high-velocity stream.
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www.cepmagazine.org July 2006
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Liquid ChromatographyProcess Design
Ask the Ex p e rt s
BOB DREAM
is director of pharmaceutical biotechnology for CH2M-Hill, Inc. (10 123 Alliance Road, Suite 300; Cincinnati, OH 45242;Phone: (513) 587-7143; Fax: (513) 530-5541;E-mail: ro b e rt . d ream@ch2m.com). Dream has 24 years of industrialexperience in process planning, cycle-time analysis, scaleup, re g u-l a t o rycompliance and asset management. He is a re n owned speak-er on the state-of-the art in biotechnology and pharmaceutical pro-cess design whose work is widely published. Dream is ap rofessional engineer and an active member of International Soc.of Pharmaceutical Engineers (ISP E ) .
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