ELSEVIER PII: SOO32-9592(97)00068-X

Process Biochemiws Vol. 33, No. 1, 47-55, 199X pp. 0 1998 Elsevier Science Ltd

All rights reserved. Printed in Great Britain 0032-9592198 $19.00+~.00

The influence of the degree of cross-linking, type of ligand and support on the chemical

stability of chromatography media intended for protein purification

Mikael Andersson, Mats Ramberg and Bo-Lennart Johansson*

Amersham, Pharmacia Biotech, 751 82 Uppsala, Sweden

(Received 5 July 1998; accepted 12 July 1997)

Abstract

The release of organic compounds from different liquid chromatography media in static conditions has been analysed with a total organic carbon (TOC) analyser. TOC results show that chemical stability increases with the degree of cross-linking in agarose beaded chromatography media and thus extend the working pH-range of the media. Of the unsubstituted chromatography media investigated, Sepharose@ 6B, Sepharose CL-6B. Sepharose 4 Fast Flow, Sepharose 6 Fast Flow and Sepharose High Performance, the latter was the most stable medium. Sepharose High Performance releases only about 0.06% of its total carbon content after I week in 0.01 M HCI. Agarose beads are more stable to basic conditions (pH 14) compared with acidic conditions (pH 2). From UV spectroscopic and gel filtration results it was found that all Sepharose media release low amounts of 5-(hydroxymethyl)-2-furaldehyde and agarose fragments in acidic conditions. To investigate the effect of different ligands on chemical stability Q Sepharose 6 Fast Flow, DEAE Sepharose 6 Fast Flow, SP Sepharose 6 Fast Flow, CM Sepharose 6 Fast Flow, Phenyl Sepharose 6 Fast Flow, Octyl Sepharose 4 Fast Flow media were also studied under static conditions. In basic conditions it was found that all these chromatography media release carbon compounds to a higher extent than the unsubstituted Sepharose support. In addition, Hofmann elimination of Q and DEAE groups contributes to the decrease in the carbon content of the corresponding anion exchangers. During exposure to acidic conditions (pH 2) the release of carbon compounds was lower than the release from the support to which the ligands were coupled. The exceptions are Octyl Sepharose 4 Fast Flow and SP Sepharose 6 Fast Flow. In the case of Octyl Sepharose 4 Fast Flow, the ligand did not seem to influence chemical stability, whereas the SP group increases the degradation of the Sepharose support. In the case of SP Sepharose 6 Fast Flow the stability in acidic conditions can be improved by increasing the ionic strength. Anion exchangers based on different support polymers (agarose-, polystyrene-, methacrylate- and polyvinyl-based matrixes) were studied under static conditions. Agarose-based anion exchanger was the most stable in basic conditions (pH 14). In acidic conditions (pH 2) the chemical stability was about the same for many different anion exchangers. 0 1998 Elsevier Science Ltd

Keywords: chromatography media, chemical stability, cross-linking, support, ligand, protein purification.

Introduction ular size, charge and hydrophobicity, is used to isolate biopolymers [l-3]. The amount of biopolymers that

Over the last 20-30 years the preparative chroma- can be separated per unit of time and the purity of the tography of biopolymers such as proteins, peptides and purified compounds are often the determining factors polynucleotides has gained ever-increasing importance. in preparative chromatography. Although these factors Normally, a purification process based on multimodal provide clear directives regarding the choice of the chromatography, which exploits differences in molec- separation medium, practical implications, such as

fouling, regeneration, chemical stability and length of *To whom correspondence should be addressed. useful life of the chromatography medium, are equally

47

48 M. Andersson et al.

important [4,5]. For example, long-term performance of chromatography media is mainly dependent on two normal events: deterioration due to irreversible adsorp- tion of contaminants, and chemical destruction resulting from harsh washing to clean or sanitize. Knowledge of the chemical stability of the packings during cleaning-in-place (CIP), sanitization-in-place (SIP) and running conditions is, therefore, of utmost importance when designing optimal chromatographic steps. Furthermore, it is possibly that degradation products from the chromatography medium could find their way into the final purified biopolymer. There is little published information available from manufac- turers about the quantity or toxicity of leached products from chromatography media [6]. Despite this, numerous chromatography media are used in processes for the production of therapeutics that have been approved by regulatory authorities.

The leakage from chromatography media can be influenced by many factors. The type of ligand, the coupling chemistry employed, the degree of cross- linking and the type of support could all affect the result. The main aim of this work was to study the release of organic compounds from Sepharose media with different ligands and levels of cross-linking. Total organic carbon (TOC) analysis was used to study the release of water-soluble organic compounds over a wide pH range. Another part of the study was focused on different anion-exchangers from six suppliers to elu- cidate differences in chemical stability of various support polymers.

All experiments were performed in static conditions with incubation times of 168 h. This experimental set-up results in a much higher concentration of leach- ables than can be expected from chromatographic con- ditions [4].

Experimental

Chemicals and apparatus

Hydrochloric acid and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Potassium hydrogen phthalate was obtained from Kanto Chemical (Japan); Nfl,W,W-tetraethylethylenediamine was from Janssen Chimica (Beerse, Belgium); ethanol (995%) was from Kemetyl (Stockholm, Sweden); 5-(hydroxy- methyl)-2-furaldehyde (HMF) was from Aldrich (Stein- heim, Germany); 2-phenoxyethanol was from Fluka (Buchs, Switzerland). The nine poly(ethylene glycol) (PEG) standards (Mr 200, 300, 400, 600, 1000, 1500, 2000, 3000, 4000 g/mol) were from Merck (Darmstadt, Germany). Sepharose@ 2B, Sepharose 4B and Sepharose 6B; Sepharose CL-2B, Sepharose CL-4B and Sepharose CL-6B, Sepharose 4 Fast Flow, Sepharose 6 Fast Flow, Q Sepharose 6 Fast Flow, DEAE Sepharose 6 Fast Flow, SP Sepharose 6 Fast Flow, CM Sepharose 6 Fast Flow, Phenyl Sepharose 6

Fast Flow, Octyl Sepharose 4 Fast Flow, Sepharose High Performance, DEAE Sephadex@ A-50, SOURCE@ 15Q and SOURCE 30Q were from Phar- macia Biotech AB (Uppsala, Sweden). The following media were also used: POROS@ 50HQ from PerSe- ptive Biosystems, Macro-Prep@ High Q from BIO-RAD, Q HyperDTM F from BioSepra, Toyo PearlTM Super Q, and Fractogel@ EMD DEAE-650 (M) from Merck.

The TOC reference material was National Institute for Standards and Technology SRM no. 917a (ICR Standards, Environmental Resource Associates, Arvada, CO, USA).

For carbon measurement in water solutions, a TOC analyser TOC-5000 with an automatic sample injector ASI- (Shimadzu, Japan) was used. For removal of carbon dioxide (CO,) from the air used in the TOC analyser, a COZ purifier (PEAK Scientific, UK) was used. High purity Mini-Q water (MQ water) was obtained from a Milli-Qls5 Plus water purifier (Milli- pore, France).

For column experiments, FPLC@ System was used (Pharmacia Biotech AB, Sweden). FPLC System con- sisted of an LCC500 control unit, a P-500 high-pre- cision pump and an MV-8 motor valve.

Analysis of the molecular size distribution of agarose released from Sepharose media was performed on an HR lo/50 column packed with Superdex@ medium (type of Superdex 30 prep grade). Column effluents were monitored by using a refractive index detector (RID-6A, Shimadzu, Japan). A Shimadzu C-R4A integrator was used to store chromatographic results.

Spectrophotometric analyses were performed on a UV/VIS Lambda 16 HP spectrophotometer (Perkin- Elmer, USA) with 10.0 mm Spectrosil@-type l-Q-10 cuvettes (Lightpath, UK). Evaluation was done with UVDM software (Perkin-Elmer, USA).

The total carbon content of dry chromatography media was analysed by Mikrokemi AB (Uppsala, Sweden). A CHNS elemental analyser EA 1108 (Fisons Instrument) was used for these measurements.

Treatment of chromatography media during static conditions in different water solutions

Incubation of chromatography media under static conditions The chromatography medium sample was first washed on a glass filter funnel (pore size, G3) with a minimum of 10 bed volumes of MQ water to ensure removal of residues of shipping buffer (i.e. 20% ethanol). The chromatography medium was sucked dry with a water pump between each washing step. Portions of about 20 g of the drained chromatography medium were transferred into a 250 ml plastic beaker and 200 ml of MQ water was added before the beaker was capped with plastic film and left at room temperature over-

Chemical stability of chromatography 49

night. The chromatography medium suspension was then transferred into a 125 ml glass filter funnel (pore size, G3) and washed 10 times with MQ water as described above. Approximately 5 g of the drained chromatography medium was transferred for ‘determi- nation of dry weight’ (see below) and 10 g of the drained chromatography medium were quantitatively transferred into a 50 ml glass filter funnel (pore size, G3). The chromatography medium was equilibrated with 2 x 25 ml of the incubation solution and sucked dry. After equilibration the chromatography medium was quantitatively transferred into an Erlenmeyer flask (100 ml) and 50.0 ml of the incubation solution was added. The flask was placed in an oven at 40°C for 1 week (168 h). After incubation, the supernatant was removed and transferred into a 50 ml Erlenmeyer flask for further analysis. The incubation solutions used for the static experiments were MQ water and solutions of hydrochloric acid (pH 2 and pH 4) or sodium hydroxide (pH 10 and pH 14).

Determination of dv weight Approximately 5 g of the drained chromatography medium was weighed into a preweighed glass filter funnel. The chromatography medium was washed with water, shrunk with acetone and finally dried at 105°C for approximately 12 h. The chromatography medium was kept in a desiccator for 1 h before weighing. The total carbon content was analysed for all dried chroma- tography media (Table 1).

TOC analysis in static experiments

TOC is a quantitative measure of the total (non-purgable) organic content of an aqueous solution. To reduce the blank signal due to carbonate, all

Table 1. Carbon content in dry Sepharose media and the dry weight of the pretreated Sepharose media

Product Carbon content

(%)”

Dry weighth

Sepharose 6B 45.0 55.8 Sepharose CL-6B 46.2 55.7 Sepharose 4 Fast Flow 47.0 44.2 Sepharose 6 Fast Flow 47.0 66.0 Sepharose HP 47.8 68.5 Q Sepharose 6 Fast Flow 44.3 159.8 DEAE Sepharose 6 Fast Flow 49.1 89.6 SP Sepharose 6 Fast Flow 41.8 137.4 CM Sepharose 6 Fast Flow 45.2 78.3 Phenyl Sepharose 6 Fast Flow 50.6 69.9 Octyl Sepharose 4 Fast Flow 48.6 43.3

“The carbon content is expressed as 100 x (gram carbon in the dry chromatography medium/gram dry chromatography medium). ?he dry weight is expressed as milligram dry gel/gram pre- treated incubated gel. See the Experimental section for details.

Table 2. Measured TOC concentrations of water solutions of different organic compounds

Compound Calculated Measured Recovery (%,)

(ppm (ppm carbon) carbon)

Ethanol 4.01 3.82 95 HMF 4.04 3.96 98 N,N,N’,N’- 4.01 3.75 94

tetraethylethylenediamine 2-Phenoxyethanol 4.09 3.75 92

samples were acidified with 2 M HCI and sparged with COz-free air for 3 min. With this procedure the TOC blank signal from 1 M NaOH was always lower than 5% of the sample signals from the static incubation experi- ments (see above). The TOC concentration was evalu- ated from a calibration graph of potassium hydrogen phthalate. Standards were prepared in the concentra- tion range 0.5-5.0 pg carbon/ml and samples were diluted with MQ water prior to analysis to this carbon content. To study the systematic errors of the TOC analysis, four suspected leakage compounds (N,NJV’,N’-tetraethylethylenediamine, ethanol. HMF, 2-phenoxy- ethanol), identified earlier from different Sepharose media [4,5,7], were analysed. Water solutions of these compounds (concentration of about 4 pg carbon/ml) were prepared and analysed as described above. The recovery of all four compounds was within the interval 92-98% (Table 2).

For method control, a reference material was also analysed after it had been diluted 10 times. The results from 16 analyses of the diluted reference solution over a period of 20 days were all within the certified interval.

In addition, triplicates were performed of all samples and standards. The relative standard deviation of these triplicates was always lower than 2%.

In order to compare the results from chroma- tography media with different carbon contents, the leakage results were also related to the total amount of carbon of the incubated chromatography media (Table 1).

Gel filtration analysis of supematants from incubated chromatography media during .static conditions

500 ~1 of the neutralized supernatants from the static experiments (see above) were injected onto the Superdex column and eluted with 0.1 M NaCl at a flow rate of 0.5 ml/min. A rough estimate of the molecular weight of the leakage compounds was evaluated from the selectivity curve (K,, versus the logarithm of the molecular weight) of the PEG standards. The total bed volume (Vc = 39.6 ml) and the void volume

50 M. Anderson et al.

(I/0 = 14.2 ml) were determined geometrically and by injection of Blue Dextran. The K,” values of the PEG standards were calculated from the equation

where I’, is retention volume of the PEG standards.

Results and discussion

Chemical stability of chromatography media

The introduction of gel filtration chromatography in 1959 as a practical separation technique [S] has resulted in a vast number of commercially available chromatography media [2,9-111. During the 35 years of development, chromatography media for preparative separations have been continuously improved. An ideal chromatography medium for preparative separations should possess the following properties: hydrophilic@ and inertness (i.e. without non-specific interaction), chemical stability under extreme conditions, high dynamic binding capacity, high mechanical rigidity, high selectivity and good batch-to-batch reproducibility. The relative importance of each of these properties depends upon the specific purification protocol and the scale of the chromatographic step to be used.

A fundamental approach to improve the chemical and mechanical stability of beaded chromatography media has been to increase the degree of cross-linking. Therefore, Sepharose media cross-linked to different degrees were tested with respect to leakage at different static conditions.

Release of carbon compounds from different unsubstituted Sepharose media in acidic and basic solutions under static conditions

TOC analysis was used to study the release of organic compounds from three different agarose-based gel filtration media in static conditions in different aqueous solutions of HCl and NaOH. From Fig. 1 it can be seen that carbon leakage was highest for Sepharose 6B and lowest for Sepharose High Perform- ance. Sepharose 6B is chemically unstable in extreme acidic and basic conditions, whereas Sepharose High Performance is stable over the whole pH interval investigated. Figure 1 also shows that Sepharose CL-6B is stable at high pH, but that it decomposes slightly in acidic conditions. The main chemical difference between these chromatography media that can explain the observed results is the degree of cross-linking, which increases in the order

Sepharose 6B < Sepharose CL-6B

< Sepharose High Performance

It should be noted that Sepharose 6B beads are stabi- lized only by hydrogen bonding and not by covalent cross-links. This explains the release of organic com- pounds from Sepharose 6B at neutral pH conditions (Fig. 1). It is well known that cross-linked agarose- based chromatography media are functionally stable after treatment in extreme alkaline solutions but that they deteriorate in acidic solutions [7]. To study more thoroughly the influence of the degree of cross-linking on the chemical stability of agarose-based chroma- tography media in acidic conditions, Sepharose 4 Fast Flow and Sepharose 6 Fast Flow were also investi- gated. These agarose gels are cross-linked to a lesser

Sepharos? 68

Sepharosp CL-6B

Sepharosp High Performance 0 2 4 6 8 10 12 14

pH of incubation solution

Fig. 1. The effect of pH on the amount of carbon released from different Sepharose media. Carbon leakage is expressed as per mil loss of carbon (1000 x total amount of released carbon/total amount of carbon in incubated chromatography medium). The chromatography media were incubated with 10W2 M HCI, 10P4 M HCI, MQ water, 10e4 M NaOH and 1.0 M NaOH at 40°C for 1 week.

Chemical stability of chromatography 51

degree than Sepharose High Performance but to higher degree than Sepharose CL-6B. The results pre- sented in Fig. 2 show that Sepharose 4 and 6 Fast Flow have a higher chemical stability than those chroma- tography media with a lesser degree of cross-linking in acidic conditions (0.01 M HCl, pH 2). Sepharose Fast Flow media release about 1% of their total carbon content after 1 week in O-01 M HCl. As expected, the highest cross-linked chromatography medium, Sepharose High Performance, is the most stable agarose bead in acidic conditions (Fig. 2).

Acid-catalysed reactions of carbohydrates are part of a very complex reaction network involving a variety of isomerization, condensation, fragmentation and dehy- dration reactions [12]. It is well established that a main decomposition product of hexoses in acidic conditions is HMF [13]. HMF in water has a characteristic UV spectrum with a major band at 285 nm (E = 16500 l/(mol cm)) and a minor band at 228 nm [14]. The peak at 285 nm was utilized to analyse the amount of HMF released during static experiments at pH 2. According to the spectrophotometric results, all the Sepharose media investigated release HMF. However, release of HMF can only explain a minor part of the TOC results. In the case of Sepharose High Performance, Sepharose 6 Fast Flow, Sepharose CL-6B and Sepharose 6B the release of HMF corresponds to only 4%, 0.2%, 0.03% and 0.07% respectively of the observed TOC release.

To clarify the influence of agarose content on HMF leakage, the same volume of the three chromatography media Sepharose CL-2B, CL4B and CL-6B were incu- bated for 1 week at 40°C in 0.01 M HCI. Fieure 3

123 -

10,o - T;; +

g - S 7,5

4

2 5,0-

9

2,5-

O--

clearly shows that the UV response increases in the order

Sepharose CL-2B < Sepharose CL4B

< Sepharose CL-6B

Sepharose CL-6B, CL-4B and CL-2B contain 6%, 4% and 2% (w/w) agarose respectively, which explains the different amounts of HMF released. The fact that HMF only explains a minor part of the TOC results indicates that polysaccharide fragments (agarose frag- ments) are released due to hydrolysis of the glycoside bonds. This was also verified with gel filtration analysis (refractive index detector) of the incubation solution (0.01 M HCI) from the Sepharose media. The results depicted in Fig. 4 show that Sepharose CL-6B releases agarose fragments with a broad apparent molecular weight distribution (400 to > 4000 g/mol). Release of agarose fragments in acidic conditions has also been verified for the other chromatography media investi- gated (results not shown).

Release of carbon compounds from different substituted Sepharose media in acidic and basic solutions under static conditions

The effect of the ligand in the release of compounds from chromatography media is related to the type of ligand, coupling chemistry, ligand density, properties of the support and experimental conditions. An optimal coupling procedure must give a covalent attachment to the support or the spacer arm. In addition, the covalent linkage must be stable in all conditions to which the

Sepharose@ media

66

6 Fast Flow

4 Fast Flow

High Performance

Increasing content of cross-linker ??

Fig. 2. The release of carbon compounds from different Sepharose media at pH 2. Carbon leakage is expressed as loss of carbon in percent (100 x total amount of released carbon/total amount of carbon in incubated chromatography medium). The chroma- tography media were incubated in lo-’ M HCl at 40°C for 1 week.

52 M. Anderson et al.

chromatography media will be exposed. There is no chromatography medium available that does not leach compounds. However, there is a broad choice of activa- tion procedures and types of ligand used in the produc-

q35

0.25

240 280 3io 360 460 Wavelength (nm)

Fig. 3. The UV response of HMF in supematant from Sepharose CL-2B (blue), Sepharose CL-4B (red) and Sepharose CL-6B (green). Equal volumes of chromatography media were incubated for 1 week at 40°C in 0.01 M HCI.

0 10 20 30 40 50 60 70 80

Re.tention the (min)

Fig. 4. Gel filtration elution pattern of agarose fragments in supernatant from Sepharose CLdB incubated for 1 week at 40°C in @Ol M HCl. 500 ~1 supernatant (adjusted to pH 7) were injected onto an HR lo/50 column packed with Superdex medium and eluted with water containing 0.1 M

NaCl at a flow rate of 0.5 ml/min. See the Experimental section for details.

tion of liquid chromatography media [10,15,16], and many of these chromatography media have not been fully documented with respect to leakage. The most widely investigated leakage process is the release of ligands coupled via cyanogen bromide activation of the support [17-191, and the stability of ligands coupled in other ways has also been studied [5-7, 18, 20-221. In certain chromatography media the ligand can shield the support from degradation. It is well known that silica-based packings for reversed phase chroma- tography are unstable in basic pH systems. However, the dissolution rate of silicates can be decreased by changing the ligand length (alkyl-chain length) or the coverage of the silica support [23,24].

To investigate the influence of the ligand on the stability of chromatography medium more thoroughly, a number of different Sepharose-based packings have been examined. Chromatography media for hydrophobic interaction, cation-exchange and anion- exchange chromatography were tested (Table 3). All media are based on Sepharose 6 Fast Flow, except for Octyl Sepharose Fast Flow which is produced with Sepharose 4 Fast Flow as support. Partial structures of the different chromatography media are depicted in Fig. 5. Under static conditions (1 week at 40°C) in sodium hydroxide solutions (pH 10 and 14) it was found that all substituted chromatography media released a higher amount of carbon compounds (expressed as per mil loss of carbon, Table 3) than the Sepharose 6 Fast Flow support. This may be due to selective cleavage of ether bonds in basic conditions, since all ligands are coupled to the Sepharose support via this type of bond (Fig. 5). Cleavage of ether bonds

Table 3. Carbon leakage from two Sepharose supports and Sepharose Fast Flow media with different types of functional group. The carbon leakage was determined in the super- natant after storage over a period of 1 week in different solutions of HCI and NaOH at 40°C

Product Carbon loss (mg/g)”

pH=2 pH=4 Hz0 pH=lO pH=14

Sepharose CL-6B 79.4 3.8 0.02 0.02 1.0 Sepharose 6 11.1 0.5 n.d. n.d. 0.1

Fast Flow Q Sepharose 6 0.2 0.1 0.2 0.1 0.8

Fast Flow DEAE Sepharose 6 0.2 O-2 0.2 0.2 5.3

Fast Flow SP Sepharose 6 63.1 0.4 O-3 0.2 3.1

Fast Flow CM Sepharose 6 1.5 0.1 0.3 0.5 2.7

Fast Flow Phenyl Sepharose 6 2.7 0.3 0.2 0.1 1.4

Fast Flow Octyl Sepharose 4 11.4 1.9 1.0 0.6 6.1

Fast Flow

“1000 x (total amount of carbon found in the incubation solu- tion (g)/total amount of carbon in incubated Sepharose medium (8)); n.d., not detected.

Chemical stability of chromatography 53

(0Cl-w C8H,-OCH&HCH2-9

;.:\

(DEW. (CH&H&NC~H~C~#-CycH,-O- ?H

/- 0-CH,CHCH20CH~H&H,-Soj (SP)

1 Sepharose FF )-0-CH2-COO’ (CM)

(DUE) (CH,Ui&N~-C~H,-O-i

v

‘~-O-C”~~CH~H,-~~Cnkcp,

0

OH

(PHENYL) OCH2bHCH2-

Fig. 5. Partial structures of different ion exchange and hydrophobic interaction Sepharose Fast Flow media. *Tandem group formed by the further derivatization of a coupled DEAE-group.

has been observed for different phenyl media [7]. The release of carbon compounds at pH 14 was highest for DEAE Sepharose 6 Fast Flow and Octyl Sepharose 4 Fast Flow. This loss corresponds to about 0.6% decrease in carbon content. In the case of DEAE Sepharose 6 Fast Flow, the loss of carbon at pH 14 (Table 3) is, to some extent, caused by the release of iVfl,WJV’-tetraethylethylenediamine due to the Hofmann elimination reaction of the tandem group [S]. In addition, the pungent fishy odour released from Q Sepharose 6 Fast Flow during the incubation experi- ment at pH 14 can also be explained by the Hofmann elimination of the Q group (Fig. 5). The odour is due to trimethylamine being released. Since the nose is exceedingly sensitive to this compound [25], very small amounts are easily detected. Despite the smell, it has previously been shown that the ion-exchange capacity of Q Sepharose High Performance is not affected signi- ficantly by long-term treatment in basic conditions [26].

During incubation in acidic conditions (pH 2 and 4), the release of carbon from Q, DEAE, CM and Phenyl Sepharose 6 Fast Flow is less than for the Sepharose support (Table 3). In contrast, treatment of SP Sepharose 6 Fast Flow at pH 2 gave a loss of carbon about six times higher than for Sepharose 6 Fast Flow (Table 3). Table 4 also shows that HMF is released to a greater extent from SP Sepharose 6 Fast Flow than from Sepharose 6 Fast Flow, Sepharose CL-6B and Q Sepharose 6 Fast Flow. All these results clearly show that the presence of SP groups (Fig. 5) increases the

Table 4. Determination of HMF in the supernatant after storage of four different Sepharose media at 40°C in 0.01 M HCI for 1 week

Product HMF Share of the concentration total amount of

carbon leakage” (&ml)

Q Sepharose 6 Fast Flow n.d. n.d. Sepharose CL-6B 0.18 0.28 Sepharose 6 Fast Flow 0.21 2.09 SP Sepharose 6 Fast Flow 1153 10%

“Expressed in per mil (%); n.d. not detected.

degradation of the Sepharose support in acidic condi- tions. It should be noted that the manufacturer does not recommend using SP Sepharose below pH 4. However, preliminary results have shown that an increase in ionic strength (addition of sodium chloride) reduces the release of carbon compounds from SP Sepharose 6 Fast Flow in acidic conditions (result not shown). This will be investigated and reported. The pH in the microenvironment around the SP groups may also play an important role here. It is known that the pH of cation exchange media is lower than the sur- rounding buffer solution due to Donnan effects [27]. This difference will be larger at low ionic strengths. The Donnan effects can probably also explain the increased stability of Q and DEAE Sepharose 6 Fast Flow over Sepharose 6 Fast Flow at acidic conditions. In the case of CM Sepharose 6 Fast Flow, the CM group (Fig. 5) has a pK, value of about 4 [28]. This contributes to the stability of this chromatography medium in acidic conditions. Table 3 also indicates that substitution of the Sepharose Fast Flow support with phenyl ligands makes it more resistant to acid decom- position than when substituted with octyl ligands. Fur- thermore, a decrease in pH from 4 to 2 resulted in a tenfold increase in carbon loss from all chroma- tography media except for Q and DEAE Sepharose 6 Fast Flow; these chromatography media were not influ- enced by pH changes in this interval (Table 3).

Release of carbon compounds from anion exchanger based on different matrixes

Nine different anion exchangers from six chroma- tography media suppliers were studied under static conditions similar to those described above. From Table 5 it can be seen that great variations in chemical stability between the different anion exchangers were observed. Information on all the matrix properties having a direct impact on the chemical stability is not available from the producers. Therefore, it is difficult to interpret the results in detail. However, it can be noted that the largest variation in carbon leakage from the anion exchangers (Table 5) was observed under basic conditions. The agarose-based anion exchange

54 M. Andersson et al.

Table 5. Carbon leakage of anion exchanger from different suppliers. The carbon leakage was determined in the supernatant after storage over a period of 1 week in 0.01 M HCl and 1.0 M NaOH at 40°C

Anion exhanger Matrix Particle size” Carbon content’ Carbon loss’ (%o)

Q Sepharose 6 Fast Flow Agarose DEAE Sephadex A-50 Dextran SOURCE 15Q Polystyrene SOURCE 30Q Polystyrene POROS 50HQ Polystyrene Q HyperD F PolystyreneISi Macro-Prep High Q Methacrylate Toyo Pearl Super Q Vinyl polymer Fractogel EMD DEAE-650 (M) Vinyl polymer

(w) (%I pH2 pH 14

45-165 40-125

15 30 50 35 50

32-65 45-90

44.3 0.2 0.8 49.4 0.3 3.1 70.6 0.8 4.0 70.9 0.3 3.5 64.9 2.0 18 12.6“ 3.0 3.2 51.4 0.1 140 51.0 6.0 25 53.6 0.2 14

“Data stated by the supplier. hThe carbon content is expressed as 100 x (gram carbon in the dry chromatography medium/gram dry chromatography medium). ‘1000 x (total amount of carbon found in the incubation solution (g)/total amount of carbon in incubated medium (g)). “The matrix contains 46% Si.

chromatography medium is the most stable at pH 14; the least stable is based on methacrylate. In contrast, in acidic conditions the carbon leakage is about the same for the exchangers based on agarose, methacrylate, polyvinyl (Fractogel EMD DEAE-650) and poly- styrene. The TOC results also indicate that there are large variations between anion exchangers based on the same matrix polymer. Parameters such as cross-linking agent and its ratio and the chemical nature of the surface modification must, therefore, be carefully opti- mized to acquire a stable anion exchanger.

Conclusions

The chromatography media investigated were degraded to different degrees, in particular under extreme acidic or basic conditions. However, chromatography media quality is continually improved by better synthesis pro- cedures. This was illustrated by the results from Sepharose, cross-linked to different degrees. The most cross-linked chromatography medium, Sepharose HP, was the most stable support. It has also been shown that a ligand coupled to the support can either increase or decrease the chemical stability. Furthermore, it has been shown that anion exchangers based on different support polymers result in great differences in chemical stability.

Documenting the chemical stability of media for liquid chromatography is normally very time-con- suming. TOC analysis is a simple method to use for an initial survey of the chemical stability of different media designed for preparative chromatography of proteins. TOC can be applied to chromatography media based on agarose, dextran, cellulose, poly- styrene, polymethacrylate and inorganic chroma- tography media with organic ligands. The main disadvantage of using the TOC technique is that no structural information on the organic compounds released is obtained.

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