A purification process scheme was developed to purify therapeutic-grade human monoclonal antibodies. It features two columns
with interchangeable chromatography steps and non-affinity resins. The purification scheme includes a cation exchange column
(CEX) and a hydrophobic charge induction (HCI) column and eliminates the need of an in-process diafiltration step. Viral clearance
from this scheme is efficient due to the ability of HCI resin to remove adventitious agents. The scheme was scaled up 1,000-
to 10,000-fold with an average overall yield >70% for a variety of antibodies. Contaminant removal and product quality from
this process are comparable to those of three-column affinity and non-affinity purification schemes.
The economics of large-scale purification of proteins is important, especially for therapeutic antibodies. Antibodies make
up a large percentage of the therapeutic biologics on the market, accounting for about 30% of total recombinant protein product
sales. Revenues for 20 approved therapeutic antibodies are estimated to be about $17 billion in 2006, and expected to be over
$22 billion in 2007.1 Costs associated with antibody-based therapies tend to be particularly high because these are expensive molecules to develop,
produce, and often require high doses.
The purification scheme is very important for both final product quality and process economics, because chromatography alone
can account for two thirds of downstream processing costs. When the products are monoclonal antibodies, the resin cost for
an affinity-capture column such as Protein A can overwhelm the raw materials costs.2
Affinity chromatography often is used as a capture step to meet purity, yield, and throughput requirements, despite the development
of advanced chromatography resins with improved performance at high flow rates and binding capacity. Traditionally, the affinity-based
capture step (Protein A or G) is followed by at least one or two other chromatography steps. In addition, one or more in-process
diafiltration steps are needed to condition the feed into the following column. Not only is Protein A resin at least four
to five times more expensive than non-affinity media, it can also have issues such as ligand leaching. In general, even if
affinity chromatography is used, adequate purity and viral clearance often are not achieved unless one or more polishing steps
By eliminating specific steps in downstream processing, productivity can be maximized while maintaining the integrity and
purity of the molecule. We describe here a flexible, interchangeable, non-affinity, two-step purification method that can
be implemented at lower cost compared to affinity-based schemes due to its simplicity and short number of steps involved.
Overall recovery is high and final product quality is equivalent to more traditional protocols.
Not only did we reduce the number of steps with the proposed purification process, but we also eliminate the need for an in-process
concentration or diafiltration (which would mean more development, optimization, scale-up, cleaning and cleaning validation,
and increased process cost while risking potential loss or modification of product). Minimizing the number of steps will cut
down the number of process components, buffers, tanks, and miscellaneous equipment. Finally, the two-column purification process
can be readily implemented into the clinical manufacturing of many antibody molecules with less initial investment on development
cost, time, and resource requirements.
TWO-COLUMN PURIFICATION PROCESS
This purification process explores the integration of the separation principles of two different resins, a cation exchange
(CEX) resin (Poros 50HS or Fractogel EMD SE HiCap) and a hydrophobic charge induction (HCI) resin (MEP HyperCel). The proposed
scheme adapts to different feed types and user implementation choices, while still clearing host cell proteins (HCPs), viral-like
particles, nucleic acids, product-related contaminants, and media additives (e.g., methatrexate, insulin, vitamins) to levels
that allow the therapeutic use of the final purified material.