B I O P R O C E S S TECHNICAL
`
`Regeneration Studies
`of Anion-Exchange
`Chromatography Resins
`
`Paul K. Ng and Valerie McLaughlin
`
`C hromatography columns can
`
`become contaminated by a
`variety of protein and
`nonprotein species during a
`purification campaign. Consequences
`of column contamination include an
`increase in backpressure, loss of signal
`resolution, altered product yield, and
`medium discoloration. Common
`chromatographic contaminants include
`• residual proteins
`• nucleic acids
`• lipids
`• endotoxins
`• viruses and bacteria
`• metal ions.
`Generally, methods for cleaning-in-
`place (CIP) and sanitization-in-place
`(SIP) of chromatographic resins are
`selected based on the interplay and
`relevance of three factors: ease of
`operation, historical experience, and
`performance requirements. In most
`cases, a column decontamination
`method chosen by a laboratory forms
`the basis not only for process validation,
`but also for subsequent scale-up. For this
`
`PRODUCT FOCUS: ALL
`BIOPHARMACEUTICALS
`
`PROCESS FOCUS: PURIFICATION
`(CHROMATOGRAPHY, CLEANING)
`
`WHO SHOULD READ: RESEARCH, PROCESS
`DEVELOPMENT, AND MANUFACTURING
`
`KEYWORDS: ANION-EXCHANGE
`CHROMATOGRAPHY, NAOH, NACL,
`ENDOTOXINS, DNA, CIP, SIP,
`QUANTITATION
`
`LEVEL: INTERMEDIATE
`
`52 BioProcess International
`
`MAY 2007
`
`reason, an ideal scenario would be
`development and use of a generic
`decontamination method. At present,
`however, even taking into account the
`varying types of resins available,
`considerable disparity of CIP and SIP
`procedures is apparent in instructions
`available from manufacturers of
`chromatography resins.
`Many traditional cleaning solutions
`are used for CIP and SIP. Table 1 lists
`target contaminants of these solutions.
`Unquestionably, the cleaning strategy
`that has attracted the most attention
`and produced the most dependable
`results is the combination of sodium
`chloride (NaCl) and sodium hydroxide
`(NaOH). It has repeatedly proven
`effective in chromatography column
`decontamination. The key advantage of
`NaOH is its bactericidal action: NaOH
`inhibits the growth of and kills many
`bacteria and microorganisms. When
`NaOH–NaCl is applied to base-resistant
`chromatography resins supplied by
`various manufacturers, it has proven to
`be highly effective in validation studies
`for removal of residual proteins, viruses,
`and endotoxins (1, 2).
`In our investigation, we addressed
`the clearance of DNA because there is
`limited relevant information or
`supporting data on mass balance.
`DNA, being highly negatively charged,
`has strong affinity for the positively
`charged surfaces of anion-exchange
`resins. Any DNA not removed by a
`cleaning procedure will gradually
`accumulate over time and diminish
`column binding capacity and
`selectivity. Accordingly, we chose to
`examine chromatography issues such as
`
`Scanning electron micrograph of the
`UNOsphere Q anion-exchange
`chromatography resin
`(WWW.BIO-RAD.COM)
`
`postcleaning DNA recovery and
`selectivity, as are discussed here. We
`also touch upon simultaneous clearance
`of endotoxin and residual proteins.
`MATERIALS AND METHODS
`DNA: For DNA recovery studies we
`used sheared salmon sperm DNA
`(catalog #9610-5-D, R&D Systems of
`Minneapolis, MN, www.rndsystems.
`com). As indicated in the manufacturer’s
`package insert, the material contains
`DNA fragments ranging in size from
`200 to 500 base pairs.
`Quantitation of DNA: Absorbance at
`260 nm (A260) was used for monitoring
`DNA concentration with a conversion
`factor of 50 µg/mL DNA per
`absorbance unit. DNA concentration
`was also measured using a dye-based
`assay with PicoGreen (Invitrogen of
`Carlsbad, CA, www.invitrogen.com),
`which fluoresces on binding to double-
`stranded DNA. After adding the
`working solution of PicoGreen reagent
`to the sample and incubating it at room
`
`Page 1
`
`KASHIV EXHIBIT 1056
`IPR2019-00791
`
`

`

`temperature for 2–5 min., we measured
`fluorescence using a Cary Eclipse
`spectrophotometer (Varian, Inc. of
`Walnut Creek, CA, www.varianinc.
`com) with excitation at 480 nm and
`emission at 520 nm. The detection limit
`of the PicoGreen assay is 250 pg/mL of
`double-stranded DNA (75 pg in a
`300 µL sample volume). Linearity, with
`a regression coefficient of >0.99, was
`routinely obtained in a standard curve
`spanning 0–500 ng/mL.
`Quantitation of Endotoxin: For
`single sample assays, we used the
`Endosafe-PTS reader (Charles River
`Laboratories of Wilmington, MA,
`www.criver.com), a point-of-use test
`system that involves existing FDA-
`licensed Limulus amoebocyte lysate
`(LAL) reagents in a test cartridge
`with a handheld spectrophotometer.
`Sensitivity of the assay is 0.05
`EU/mL.
`UNOsphere Q Chromatography
`Support: All chromatography
`experiments were conducted using
`UNOsphere Q support packed in Bio-
`Scale MT2 or MT10 columns (Bio-
`Rad Laboratories, Inc. of Hercules,
`CA, www.bio-rad.com). The MT2
`column dimensions are 0.7 cm in
`diameter and 2.6–5.2 cm high. The
`MT10 column dimensions are 1.2 cm
`in diameter and 8.8 cm high.
`Endotoxin Concentrate: The
`endotoxin concentrate consisted of
`equal amounts of lipopolysaccharides
`from Escherichia coli, Salmonella
`enterica serotype abortus equi, and
`Pseudomonas aeruginosa 10 (all
`purchased from Sigma-Aldrich, St.
`Louis, MO, www.sigma-aldrich.com).
`It was assayed with the Endosafe-PTS
`reader and determined to have 6.64 ×
`106 EU/mL.
`Selectivity: A Bio-Rad Laboratories
`protein standard for anion-exchange
`chromatography (catalog #125-0561),
`comprising equine myoglobin,
`conalbumin, chicken ovalbumin, and
`soybean trypsin inhibitor, was
`separated using a gradient method
`(buffer A, 20 mM Tris, pH 8.5; buffer
`B, 20 mM Tris, 1.0 M NaCl, pH 8.5).
`The retention time of each protein was
`determined from the chromatogram.
`Chromatography System: All
`chromatography experiments were
`
`54 BioProcess International
`
`MAY 2007
`
`Table 1: Traditional cleaning solutions for specific contaminants
`
`Cleaning Solutions
`
`1–3 M NaCl, 1–2 M NaOH
`
`Guanidine hydrochloride
`
`Contaminants Removed
`
`Residual proteins, DNA
`
`Residual proteins, lipids
`
`Urea, ethanol, isopropyl alcohol
`
`Residual proteins, lipids
`
`1–2 M NaOH, tri(n-butyl)phosphate/Tween
`
`Viruses, endotoxins
`
`Citric acid, EDTA
`
`Metal ions
`
`Table 2: Effect of wash sequence on DNA
`recovery
`
`Table 3: Measured DNA clearance using the
`PicoGreen assay
`
`Process Step
`
`Flowthrough with wash
`
`Eluted fractions at 0.1 M NaCl
`
`Eluted fractions at 0.5 M NaCl
`
`Eluted fractions at 1.0 M NaCl
`
`Eluted fractions at 2.0 M NaCl
`
`2.0 M NaCl with 1.0 M NaOH wash
`
`1.0 M NaOH wash
`
`0.02 M NaOH wash
`
`Cumulative
`
`Percentage
`Yield
`
`0
`
`0
`
`0
`
`35.4
`
`10.8
`
`37.8
`
`7.8
`
`0
`
`91.8
`
`automated and performed using a
`BioLogic DuoFlow Maximizer
`chromatography system and software
`(Bio-Rad Laboratories, Inc.). Flow
`rates of the columns were maintained
`at 300 cm/hr throughout all
`experiments.
`RESULTS AND DISCUSSION
`DNA Recovery: The standard column
`hygiene sequence developed during
`this investigation is
`Clean: 2.0 M NaCl, three column
`volumes
`Sanitize: 1.0 M NaOH, three
`column volumes
`Store: 0.02 M NaOH, three column
`volumes.
`This decontamination sequence
`was studied with fractions collected
`from stepwise elution of increasing
`NaCl concentrations up to 2.0 M. A
`0.15-µg sample of salmon DNA was
`injected into a 1-mL UNOsphere Q
`column. Table 2 shows the percentage
`yield (the ratio of A260 recovery
`relative to A260 injection).
`The tested range of NaCl
`concentrations shown in Table 2 is a
`commonly used diagnostic elution
`zone for many proteins of research
`interest. These data show that
`
`Sample
`
`Feed
`
`Fractions from
`0 to 0.5 M NaCl
`
`DNA remaining
`in column
`
`Total
`DNA (ng)
`
`Percentage
`Remaining
`
`147,500
`
`417
`
`147,083
`
`100
`
`0.28
`
`99.7
`
`Table 4: Endotoxin removal from UNOsphere
`Q column
`
`Total
`Challenge
`Reduction
`(EU)
`
`Total in
`Eluate
`(EU)
`
`Percentage
`Removal
`
`Log
`Value
`
`3.3 × 105
`
`<0.16
`
`>99.999
`
`>6
`
`insignificant clearance was obtained
`across the fractions from 0 to 0.5 M
`NaCl using anion-exchange
`chromatography. Because there was no
`detectable absorbance at 260 nm in
`these fractions, their DNA fractions
`were determined with the PicoGreen
`assay. These results (Table 3) indicated
`significant DNA clearance and agree
`with data published previously (2).
`Endotoxin Clearance: Subsequent to
`soiling with a challenge of 3.3 × 105
`units of endotoxin, the column was
`washed in sequence with 2.0 M NaCl
`and 1 M NaOH. After holding in
`1.0 M NaOH for three hours, the
`column was washed with 0.02 M
`NaOH. The wash solution was
`neutralized with phosphate buffered
`saline before the LAL assay. Results of
`the experiment (Table 4) demonstrated
`excellent clearance of endotoxin
`resulting from a combination of
`removal due to charge difference and
`inactivation of residual endotoxin
`bound to the column. These data are
`consistent with the most frequently
`used CIP–SIP protocols that use
`NaOH as the sanitizing agent. A
`clearance factor of more than six orders
`of magnitude was reached; however,
`this exceptional efficiency is restricted
`
`Page 2
`
`

`

`1 mL column was scaled up to 10 mL,
`and the following cycles were repeated
`after each run: 2.0 M NaCl wash,
`1.0 M NaOH for >3 hr, and storage at
`0.02 M NaOH for >16 hr. We then
`measured the protein’s retention time,
`which is the time between injection
`and the appearance of the peak
`maximum. As shown in Figure 2,
`selectivity remained constant over 10
`cycles in a duration of 30 days. The
`data are consistent with the superior
`base stability reported previously (5).
`GOOD CLEARANCE
`A cleaning cycle using 2.0 M NaCl
`and 1.0 M NaOH has been shown to
`give good chromatographic clearance
`of DNA and endotoxin. Excellent base
`stability of the anion-exchange
`support was evidenced by no change
`in its selectivity. This regeneration
`protocol appears to be suitable for
`both validation and scale-up.
`ACKNOWLEDGMENT
`The SEM was provided courtesy of Dr. Danni
`Wang of Bio-Rad Laboratories.
`
`REFERENCES
`1 Conley L, McPherson J, Thommes J.
`Validation of the ZEVALIN Purification
`Process: A Case Study. Process Validation in
`Manufacturing of Biopharmaceuticals. Rathore
`AS, Sofer G, Eds. CRC Press: Boca Raton,
`FL, 2005; 469–521.
`2 Dasarathy Y. A Validatable Cleaning-
`In-Place Protocol for Total DNA Clearance
`from an Anion-Exchange Resin. BioPharm 9(8)
`1996: 41–44.
`3 Jungbauer A, Lettner H. Chemical
`Disinfection of Chromatographic Resins, Part
`1: Preliminary Studies and Microbial Kinetics.
`BioPharm 7(5) 1994: 46–56.
`4 Boschetti E, et al. Concerns and
`Solutions for a Proper Decontamination of
`Chromatographic Packings. Chimica Oggi
`11(3–4) 1993: 29–35.
`5 Franklin S, et al. UNOsphere Q
`support technical data, Bio-Rad Bulletin 2729
`
`(2002). 
`
`Corresponding author Paul K. Ng is senior
`staff scientist of process applications at
`Process Chromatography Division, Bio-Rad
`Laboratories, 6000 James Watson Drive,
`Hercules, CA 94547, 1-510-741-4839,
`fax 1-510-741-4114, paul_ng@bio-rad.com.
`Valerie McLaughlin is senior product
`manager of marketing at Bio-Rad
`Laboratories.
`
`Figure 1: Selectivity before and after CIP
`
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`Figure 2: Selectivity of a 10-mL UNOsphere Q column before and after CIP
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`to artificially high endotoxin feed
`concentration. At feed concentrations
`significantly lower than the challenge
`used in this case, which is a more
`common endotoxin contamination
`level, total endotoxin clearance is
`anticipated with NaOH inactivation.
`The results are consistent with data
`previously published (1).
`Residual Contaminant Clearance:
`Following CIP/SIP and between runs,
`small amounts of residual proteins,
`microorganisms, and endotoxins could
`still be present. Their concentration
`will differ among various process
`applications and is a strong function of
`feed stream. It is necessary to use
`practical methods to quantify such
`residual materials.
`Three tests, including A280,
`microbial load, and LAL, can be used
`to verify that the resin is consistently
`meeting necessary and achievable
`acceptance criteria. The process
`developer simply compares initial and
`eluted buffer values to determine the
`magnitude of residual contaminants.
`
`With regard to column sanitization,
`NaOH is generally accepted as an
`effective cleaning agent to provide
`elevated levels of cleanliness for
`chromatographic materials and other
`product contact surfaces. Its ability to
`inactivate significant levels of
`commonly found microorganisms has
`been verified (3, 4). Hence, we made
`no attempt to carry out microbial
`challenge tests in the current study.
`Selectivity of Column Before and
`After CIP: By using the recommended
`conditions (see DNA Recovery
`section), we studied the effect of CIP
`on selectivity. The data in Figure 1
`show that column selectivity was
`unaffected by the decontamination
`treatment. The slight difference in A280
`signal was due to minor variation in the
`sample load. As would be expected, any
`change must be carefully monitored
`over the lifetime of a column. Process
`developers would address such a study
`before scale-up.
`To evaluate the sanitization–
`decontamination method further, the
`
`56 BioProcess International
`
`MAY 2007
`
`Page 3
`
`