MabSelectSuRe

MabSelect SuRe? (Superior Resistance) is a member of the MabSelect? family* of affinity chromatography media for the

capture of monoclonal antibodies (MAbs) at process scale. MabSelect SuRe is composed of a rigid, high-flow agarose matrixand alkali-stabilized protein A-derived ligand. This ligand provides greater stability than conventional protein A-based mediain the alkaline conditions used

in cleaning-in-place (CIP) protocols. The enhanced alkali stability of MabSelect SuRe improves process economy; cleaningcan be performed with cost-effective reagents such as sodium hydroxide, which improves process economy and productquality.

Key performance characteristics of MabSelect SuRe are:? Novel, alkali-stabilized protein A ligand allows the use of 0.1–0.5M sodium hydroxide for CIP.

Improves product quality and reduces overall costs. Eliminates the need for expensive and corrosive CIPreagents.

High dynamic binding capacity (DBC) reduces process time and amount of medium used. High-flow agarose matrix allows processing of large volumes of feed.High stability in alkaline conditions

The MabSelect SuRe ligand was developed by protein engineering of one of the IgG-binding domains of Protein A. Aminoacids particularly sensitive to alkali were identified and substituted with more stable ones. The final construct is a tetramer ofthe engineered domain with a C-terminal cysteine, which enables single-point attachment to the matrix.Fig 1. MabSelect

SuRe is ideal for purification of large volumes of monoclonal antibodies and is resistant to cleaning- and sanitization-in-placeusing alkali.GE Healthcare

Data File 11-0011-65 AB Affinity chromatography MabSelect SuRe

Alkali-stabilized protein A-derived medium for capture of monoclonal antibodiesThe ligand is produced by validated fermentation anddownstream processes and the entire production processis free of components of mammalian origin. The resultinghighly purified ligand is immobilized to the agarose matrixthrough a chemically stable thio-ether linkage.

MabSelect SuRe is stable under alkali conditions and hasbeen tested for up to 200 cycles of CIP using 0.1 M NaOH.The combination of low ligand leakage and high DBCtogether with the high-flow matrix makes MabSelect SuRe

ideal for the purification of MAbs at process scale.

* The MabSelect family of media for process-scale purification of monoclonal antibodies comprisesMabSelect, MabSelect Xtra?, and MabSelect SuRe. MabSelect is designed for high-throughputpurification of monoclonal antibodies from large volumes of feed. MabSelect Xtra is designed formaximum DBC, which allows capture of high expression levels of MAbs from feedstock. For moreinformation on MabSelect and MabSelect Xtra, refer to data files 18-1149-94 and 11-0011-57 respectively.im

Data File 11-0011-65 AB 2

Rigid, highly cross-linked matrix allows high flow rates

MabSelect SuRe has been developed from the same rigid, highly cross-linked agarose matrix used for MabSelect. Thematrix of MabSelect SuRe allows the use of higher flow rates in process-scale purifications of Mabs compared with

conventional cross-linked agarose of similar porosity.The characteristics of MabSelect SuRe are summarized in Table 1.High dynamic binding capacity after numerous CIP cycles

Cleaning-in-place is an essential step in the production of pure Mabs in industrial applications. The main drawback withusing sodium hydroxide for CIP of conventional protein A-based media is the sensitivity of native and recombinant Protein A(rProtein A) to alkaline conditions. MabSelect SuRe, however, retains dynamic binding capacity after repeated CIP cycleswith 0.1–0.5 M NaOH.

Figure 2 shows DBC (10% breakthrough) of polyclonal human IgG (h IgG) as a function of exposure to alkaline conditions.MabSelect, with its conventional rProtein A ligand, was used for comparison. Approximately 85–90% of the initial dynamicbinding capacity of MabSelect SuRe is retained after numerous CIP cycles with sodium hydroxide.

The dynamic binding capacity of MabSelect SuRe remains high after CIP in conjunction with purification of humanized IgG 1and IgG 4 from clarified cell culture (Table 2). DBC was retained to approximately 85% of the initial binding capacity. Therecovery of both MAbs was consistently over 95%.

The dynamic binding capacity of MabSelect SuRe remains high after CIP in conjunction with purification of humanized IgG 1and IgG 4 from clarified cell culture (Table 2). DBC was retained to approximately 85% of the initial binding capacity. Therecovery of both MAbs was consistently over 95%.Table 1. Characteristics of MabSelect SuReLigand Alkali-stabilized protein

A-derived (E. coli )Ligand coupling method Epoxy activationMatrix

Rigid, highly cross-linked agarose Average particle size (d 50V )* 85 µm

Dynamic binding capacity ? At least 30 mg human IgG/ml

medium at 2.4 min residence time Recommended mobile 100–500 cm/h

phase velocity ?Chemical stability Stable in all aqueous buffers commonly used in protein Achromatography. pH working range 3–12

Cleaning-in-place stability 0.1–0.5 M NaOH Delivery conditions20% ethanol

* d 50V is the median particle size of the cumulative volume distribution.

Determined at 10% breakthrough by frontal analysis at a mobile phase velocity of 250 cm/h in a column with a bed height of10 cm.

Determined in a BPG 300 column, bed height 20 cm, operating pressure less then 2 bar.

No. of CIP cycles

D B C a t 10% b r e a k t h r o u g h (p o l y c l o n a l h I g G ) [%]1008060402000

20 40 60 80 100 120 140 160 180 200 220

Fig 2. Dynamic binding capacity of MabSelect SuRe and MabSelect for polyclonal human IgG after CIP with 0.1–0.5 MNaOH for up to 200 cycles.

Table 2. Effect of CIP cycles using 0.1 M NaOH on the DBC of MabSelect SuRe in the purification of IgG 1 and IgG 4.Antibody CIP (no. ofDBC (dynamic bindingcycles × duration capacityin minutes)[% of initial DBC])IgG 1 150 × 15 min ≥ 85%IgG 4100 × 15 min≥ 85%

Data File 11-0011-65 AB 3

Increased residence time increases dynamic binding capacity

The already high dynamic binding capacity of MabSelect SuRe is further improved by increasing sample residence time onthe medium. With a residence time of 2.4 min, the dynamic binding capacity at 10% breakthrough of humanized IgG 1,humanized IgG 4, and polyclonal h IgG is ≥ 30 mg/ml (Fig 3). Increasing the residence time to 4.8 min increases thedynamic binding capacity of the humanized immunoglobulins and h IgG to more than 38 mg/ml.h IgG 1h IgG 1

4035302520151050IgG 1IgG 1IgG 4IgG 1IgG 1IgG 42.4 minutes3.0 minutes4.8 minutes

Fig 3. Dynamic binding capacity of MabSelect SuRe as a function of residence time of the protein on the medium.Low ligand leakage

The level of leakage of the MabSelect SuRe ligand during elution is low. A normal range of leakage is estimated to be 5–20ppm (ng ligand/mg IgG). However, leakage is affected by chromatographic running conditions and the composition of thefeedstock.

Figure 4 shows the ligand leakage from MabSelect SuRe over 100 cycles of purification of a monoclonal antibody fromclarified cell culture. Fractions collected from the

purification were analyzed by noncompetitive ELISA. Figure 4 confirms the low leakage of the MabSelect SuRe ligand overnumerous purification/CIP cycles.Concentration of leached ligand

(ng ligand/mg IgG)Purification/CIP cycles12.010.08.06.04.02.00.0

Fig 4. Ligand leakage (ppm) of MabSelect SuRe over 100 cycles of CIP in conjunction with the purification of humanized IgG4. Cleaning-in-place was performed with 0.1 M NaOH and the contact time was 15 min/cycle.Low risk of host cell protein contamination or carryover

Rigorous CIP or sanitization-in-place with sodium hydroxide reduces the risk of both contamination from host cell proteinsand microbial growth in the medium, as well as carryover in the product pools.

Figure 5 is a Western blot of humanized IgG 4 fractions eluted in the ligand-leakage study described above. The Westernblot confirms that no contamination from host cell proteins or carryover is detected after 50–100 purification cycles on

MabSelect SuRe. Comparison of the separation of host cell protein standard in lane 4 with the purified IgG 4 in lanes 6–17obtained from 100 affinity purifications on MabSelect SuRe indicates no presence of the host cell proteins in the bound

fraction eluates. The absence of protein bands in lanes 18 (carryover after 50 cycles) and 19 (carryover after 100 cycles)indicates no carryover or cross-contamination between two consecutive purification cycles with an intermittent CIP cycle.Sample, data, and image were supplied by kind courtesy of Lonza Biologics plc, Slough, U.K.Data File 11-0011-65 AB 4

Comparison of MabSelect SuRe with rProtein A-based media

Figures 6 and 7 show the performance of MabSelect SuRe and MabSelect in the purification of humanized IgG 1 and IgG 4from HCCF (host cell culture fluid). Selectivity and specificity of the two media were comparable.

In a separate study, the purification of humanized IgG 4 expressed in cell culture fluid using MabSelect SuRe was comparedto the purification performance of MabSelect and MabSelect Xtra. Purification performance of the three media was similarand contamination of host cell proteins was minimal as seen on the Western blot in Figure 8. Sample, data, and image weresupplied by kind courtesy of Lonza Biologics plc, Slough, U.K.

Fig 5. Western blot of humanized IgG 4 from 1–100 purification/CIP cycles on MabSelect SuRe. The Western blot wasdeveloped with two sets of

polyclonal antibodies; one raised against the host cell proteins and another raised against the MAb. The presence of bovineserum albumin (BSA) and IgG in lane 2, as the negative and positive controls respectively, marks the specificity of the twosets of antibodies.

Lane 1: HMW marker Lane 2: BSA IgG standard Lane 4: Host cell protein

Lane 6: Purification/CIP cycle 1Lane 7: Cycle 5Lane 8: Cycle 10Lane 9: Cycle 20Lane 10: Cycle 30Lane 11: Cycle 40Lane12: Cycle 50Lane 13: Cycle 60Lane 14: Cycle 70Lane 15: Cycle 80Lane 16: Cycle 90Lane 17: Cycle 100Lane 18: Carryover after 50 cycles Lane 19: Carryover after 100 cycles Lane 20: Start material

Fig 6. Similar desorption characteristics of MabSelect SuRe and MabSelect in the purification of humanized IgG 1 fromHCCF.

Column: Tricorn ? 5/100Sample:

8 mg humanized IgG 1 in HCCF

Equilibration buffer: 20 mM phosphate, 150 mM NaCl, pH 7.4Elution buffer: 100 mM citrate, pH 3.0System: ?KTAexplorer ?10

Fig 7. Similar desorption characteristics of MabSelect SuRe and MabSelect in the purification of humanized IgG 4 fromHCCF.

Column: Tricorn 5/100Sample:

5 mg humanized IgG4 in HCCF

Equilibration buffer: 20 mM phosphate, 150 mM NaCl, pH 7.4Elution buffer: 100 mM citrate, pH 3.0System:KTAexplorer 10M r x 10320011697653121146Lane

12467

8 9 10 11 12 13 14 15 16 17 18 19

20

mAU 300025002000150********005.010.015.020.025.030.0ml

MabSelect SuRe MabSelectmAU 1500100050005.010.0

15.0 20.0 25.0 30.0 35.0 mlMabSelect SuRe MabSelectpH

Data File 11-0011-65 AB 5

Fig 8. Western blot confirming the purity of humanized IgG 4 purified on MabSelect SuRe, MabSelect, and MabSelect Xtra.

Fig 9. Pressure/flow profile for MabSelect SuRe in a packed bed (bed height 20 cm) in a BPG 300 column (i.d. 300 mm).OperationPurification

MabSelect SuRe offers high selectivity, which renders efficiency related parameters such as sample load, flow rate, beadsize and bed height less important for resolution.

The primary aim of method optimization is to establish the conditions that will bind the highest amount of target molecule, inthe shortest time, and with the highest product recovery. The degree to which IgG binds to protein A varies with respect toboth origin and antibody subclass. In general, however, all human or humanized antibodies, except for subclass 3, have ahigh affinity for protein A.Typically, the clarified feedstock is loaded onto the column directly. After sufficient washing, the Mabis normally eluted at pH 3–4.Cleaning and sanitization

Use of 0.1–0.5 M NaOH is recommended for cleaning and sanitization. Optimization of contact time, concentration, andfrequency of CIP cycles is required to achieve the best possible results.Storage

Recommended storage solutions for MabSelect SuRe are 20% ethanol or solutions containing 2% benzyl alcohol. Therecommended storage temperature is 4–8 °C.Recommendations for column packing, cleaning and

sanitization, method design, and optimization can be found in the instructions delivered with each pack of medium.Scale-up

After optimizing the antibody purification at laboratory scale, the process can be scaled up by keeping the residence timeconstant in order to maintain capacity. This can be achieved by increasing the column diameter, and keeping the mobilephase velocity and sample-to-bed volume ratio constant.

MabSelect SuRe is based on the same matrix as MabSelect and has similar pressure and flow characteristics. Thepressure/flow curve shown in Figure 9 is for MabSelect SuRe packed in a large-format BPG ? column. For more details onpacking in Chromaflow ? columns, see Application Note 11-0007-52.M r x 10320011697653121146Lane24681011

12

Lane 2: MabSelect eluate

Lane 4: MabSelect SuRe eluate Lane 6: MabSelect Xtra eluateLane 8: Antibody reference marker Lane 10: HMW markersLane 11: Host cell protein standard Lane 12:

BSA/IgG standards0.00.10.20.3800600400200

0Pressure (MPa)

M o b i l e p h a s e v e l o c i t y (c m /h )Ordering InformationProductPack sizeCode No

MabSelect SuRe 25 ml 17-38-01 25 ml 17-38-01 200 ml 17-38-02 1 l 17-38-03 5 l 17-38-04

10 l

17-38-05Lab-scale columns XK 16/20 column 18-8773-01 XK 16/40 column

18-8774-01 Packing connector XK 16 18-1153-44 XK 16/20 tube 19-0315-01 AK 16 adapter 18-8778-01RK 16/26 reservoir 18-8793-01Prepacked columns MabSelect SuRe,Tricorn 10/100 GLInquire

Related literatureData FilesBPG columns

18-1115-23 BioProcess columns 18-1167-76Chromaflow columns18-1138-92

Application notes MabSelect SuRe - Leakage 11-0011- and ToxicityMabSelect - Column packing 11-0007-52

All bulk media products are supplied in suspension in 20% ethanol. Foradditional information, please contact your local GE Healthcare representative.Recommended columns

MabSelect SuRe can be used together with most equipment available for chromatography from laboratory scale toprocess scale. To ensure best performance at process scale, pack MabSelect SuRe to a bed height of 10–30 cm.Recommended columns are listed in Table 3.Column* Inner diameterBed volume Bed height(mm) (cm)Lab scaleXK 16/4016 16–60 ml 8–30Production scale

BPG 100–300 0.8–21 l 10–30BioProcess 100–1200 0.8–339 l 10–30Chromaflow400–200013–942 l10–30

All large-scale columns can be supplied as variable bed height columns. Note that all bed height and diameter combinationsabove are possible but not necessarily suitable. For example, do not choose large-diameter columns if the bed height is low.Another general recommendation is not to choose a bed height above 30 cm.

Table 3. Recommended columns for MabSelect SuRe. For maximum productivity and robust performance, bed heights of10–30 cm are normally used

GE, imagination at work, and GE monogram are trademarks of General Electric Company.?KTAexplorer, BioProcess, BPG,

Chromaflow, MabSelect, MabSelect SuRe, MabSelect Xtra, Tricorn, and Drop design are trademarks of GE Healthcarecompanies. All third party trademarks are the property of their respective owners.? 2007 General Electric Company—Allrights reserved First published Nov. 2004

Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues mayrequire a license under US pat 5,284,933 and US pat 5,310,663, including corresponding foreign patents (assigne: HoffmanLa Roche, Inc).

All goods and services are sold subject to the terms and conditions of sale of

thecompany within GE Healthcare which supplies them. A copy of these terms and conditions is available on request.Contact your local GE Healthcare representative for themost current information.

GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UKGE Healthcare Bio-Sciences Corp., 800 Centennial Avenue, P.O. Box 1327, Piscataway,

NJ 08855-1327, USA GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, GermanyGE Healthcare Bio-Sciences KK, Sanken Bldg., 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo, 169-0073 Japan

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11-0011-65 AB 11/2007imagination at work

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