Dr. Dirk Sievers, Marketing Manager CE Pall GmbH Life Sciences, Dreieich BioPharmaceuticals Central Europe
Dr. Sylvio Bengio Marketing Chromatography and Scientific Communications Pall Life Sciences
New Sorbents and Membranes for Process Chromatography
In many biopharmaceutical companies, chromatographic purification in downstream processing is a current key focus for optimization studies. The objective is to streamline the process, which may be achieved by the elimination of intermediate unit operations. The main question, therefore, is how to optimize the adjustment of individual chromatographic process steps?
Currently there are a broad range of next-generation sorbents and membranes commercially available for process chromatography. These new chromatography media often are characterized by significantly improved performance compared to their classical predecessors. Higher dynamic binding capacities, higher operational flow rates, and specific and distinctive retention mechanisms are examples of such criteria. Nevertheless, traditional media (e.g., based on agarose or polymers developed in the 1970s or even before) continue to be routinely used for new method development due to their proven suitability for protein purification in numerous Food and Drug Administration (FDA) or European Medicines Agency (EMA) approved applications. In these cases, the potential of modern ion exchangers and novel mixed-mode or multi-mode sorbents still remains poorly exploited. As a result, significant additional costs in production may occur and result in severe economic consequences. Practical experience shows that the optimization of individual process steps (capture, purification and polishing) already offers important cost reductions; but, it is mainly through a combination of all sorbent- and membrane-based unit operations deployed in downstream processing that opens the door for effective process cost savings.
Ion Exchange Chromatography
Anion and cation exchangers have been among the most important tools in biotechnological protein purification for decades. Ion exchange chromatography is well known by regulatory authorities and constitutes a powerful and user-friendly technique suitable for all chromatographic purification steps in downstream processing. However, recently modified manufacturing processes for these sorbents have significantly improved their performance. An example is Pall’s new Q HyperCel™ and S HyperCel ion exchange sorbents, which were designed specifically as potent high capacity solutions with differentiated selectivity for the purification of antibodies and other therapeutical proteins. They are perfectly suited for biotechnological purification schemes in laboratory, pilot and full manufacturing scales.
Q HyperCel and S HyperCel sorbents are based on a robust and scalable cross linked cellulose matrix. They exhibit high dynamic binding capacities at high flow rates (and low residence times such as 1 or 2 minutes) in routine operations (Figure 1). Q and S HyperCel sorbents enable key requirements of increased throughput and optimized process economics by means of increased productivity levels. Large volumes of feedstocks can be processed quickly, and the risk of protein degradation by proteolytic degradation is also minimized. Both ion exchangers are characterized by low non-specific binding, and chemical and mechanical robustness. Selectivities and salt sensitivities differ significantly from other ion exchange sorbents and provide users with new solutions to solve challenging separations.
Dynamic binding capacities (DBC; 10% breakthrough).
a: Q HyperCel sorbent.
b: S HyperCel sorbent.
The upper diagrams document the influence of residence time on DBC.
Sample: BSA in 50 mM Tris-HCl, pH 8.4 (Q HyperCel sorbent), human IgG in 50 mM sodium acetate, pH 4.7 (S HyperCel sorbent).
Column: LRC glass column, 1.0 x 10 cm. The plots below showing DBC as a function of buffer conductivity and pH (at 2.5 min residence time) differ significantly from those of other ion exchangers, particularly in the case of cation exchangers, which offer new selectivities for method development.
Mixed-Mode (or Multi-Mode) Chromatography
New opportunities for biopharmaceutical production are also provided by sorbents that interact with the target protein by a multiple retention mechanism. Mixed-mode MEP HyperCel, HEA HyperCel, and PPA HyperCel sorbents’ binding mechanism is primarily a combination of hydrophobic and/or pseudo affinity interactions with the target molecule. Typically this is achieved without additional modification of the feedstock conductivity. Method development benefits from new and valuable selectivities for protein purification due to the differences in the retention mechanism. Sorbent screening can be easily performed using AcroWell™ or AcroPrep™ 96-well filter plate formats or ready-to-use prepacked Pall PRC columns of 1 mL or 5 mL for scale-up/scale-down studies.
MEP HyperCel sorbent is uncharged under typical loading conditions (~pH 7), whereas the majority of proteins carry a net negative charge. Under acidic conditions, both MEP ligands (pKa 4.8) and bound proteins take on a net positive charge (pI dependent). Elution is therefore induced after a moderate decrease in pH by electrostatic repulsion of positive charges of the MEP ligand and the target protein.
HEA HyperCel and PPA HyperCel sorbents carry a partial positive charge during typical loading conditions (~pH 7). The decrease in pH below the pI of the target molecule reverses protein charge from net negative to net positive, and therefore a similar electrostatic repulsion mechanism as with the MEP ligand. When the extent of electrostatic repulsion increases above the strength of hydrophobic/pseudo affinity interactions, the target molecule elutes from the column. Typically, pH 4.5 to pH 4.0 is sufficient to induce this process even if lower pH (e.g., pH 3.0) may be required in some cases. Such comparatively mild elution conditions promote the preservation of the biological integrity of the target protein. Recently, process developers have shown that the addition of 0.1 – 0.5 M arginine to MEP HyperCel sorbent elution buffers allows for protein elution at pH values around neutrality (Arakawa et al., 2009).
Sorbent Selectivity Screening
Method development at laboratory scale primarily is used to determine the optimal sorbent for a specific purification. In general, prepacked columns or 96-well plates are used for this purpose. All HyperCel sorbents are available in prepacked PRC columns for sorbent screening with volumes of either 1 mL or 5 mL. The combination of two of the larger 5 mL columns in series results in a bed height of 20 cm, which simulates typical process scale conditions.
Two-Step Antibody Purification
Monoclonal antibody purification traditionally involves a protein A capture step followed by two further orthogonal chromatographic steps [generally, a combination of two steps from any of the following: ion exchange (anion and cation), hydrophobic interaction, or sometimes hydroxyapatite chromatography]. According to the specific antibody, the sequence of post protein A steps may vary, but generally intermediate unit operations like diafiltration for buffer exchange, or addition of salt for hydrophobic interaction, are required.
An alternative two-step purification process is based on the use of MEP HyperCel sorbent for the primary capture and S HyperCel sorbent for the second chromatographic step. These two high performance sorbents are complementary to each other and assure both yield and purity of the target protein to meet the challenging demands of modern purification schemes. Requirements for intermediate unit operations are eliminated, reducing operator time, consumable costs and enhancing process economics.
Typically, the capture and intermediate chromatographic steps are in positive mode, i.e. the target molecule is selectively bound to the sorbent. However, to ensure the remaining level of contaminants (DNA, HCP, endotoxin and virus) meets regulatory approval the final chromatographic step tends to be in negative mode. In this mode, only the contaminants are removed, and the target molecule is in the flow through. Chromatography membrane adsorbers are ideally suited for negative mode or polishing applications. A significant advantage of membranes over classical sorbents is the larger pore size which allows enhanced accessibility for macromolecules (e.g., DNA and virus) and allows for faster processing using much higher flow rates than conventionally packed bed resins. Membrane adsorbers are available in a ready-to-use disposable capsule format that eliminates the need for column packing and cleaning. This reduces process times, hardware, and reagent and labor costs, and enhances process economics. The new family of Mustang XT membrane adsorbers with scalable formats meets all requirements from laboratory scale to process scale applications.
A reproducible and robust manufacturing process under GMP conditions requires high performance columns (Figure 2) and control skids to ensure the performance of the validated chromatographic process meets the desired product specifications. Flexible and robust Resolute columns and packing stations in combination with PK systems for process-scale chromatography and PKP systems for pilot-scale chromatography offer the optimal hardware platform for all types of sorbent or membrane-based chromatographic unit operations. Resolute and PK/PKP systems enable end users a smooth transition of their chromatographic separations from process development to production scale.
Chromatography columns for use in laboratory scale, pilot scale and process scale.
a: PRC prepacked columns (1 and 5 mL).
b: LRC glass columns (up to 900 mL).
c: Resolute columns (up to 300 L).
d: Resolute columns (up to 1,500 L).
Authors Dr. Dirk Sievers Marketing Manager CE Pall GmbH Life Sciences, Dreieich BioPharmaceuticals Central Europe Tel.: +49/6103/307-582 Fax: +49/6103/307-295 firstname.lastname@example.org Dr. Sylvio Bengio Marketing Chromatography and Scientific Communications Pall Life Sciences, Cergy France Tel.: +33/1/3420-7823 email@example.com www.pall.com/biopharm