High-performance size-exclusion chromatography of recombinant derived proteins and aggregated species

Journal of Chromatography, 436 (1988) 289-298 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CHROM. 20 156

HIGH-PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY OF RE- COMBINANT DERIVED PROTEINS AND AGGREGATED SPECIES

ERIC WATSON* and WILLIAM C. KENNEY

AMGEN, 1900 Oak Terrace Lane, Thousand Oaks, CA 91320 (U.S.A.)

(First received May 5th, 1987; revised manuscript received October 27th, 1987)

SUMMARY

The chromatographic behavior of some recombinant derived proteins and aggregated species was studied using high-performance size-exclusion chromatography (HPSEC). At neutral pH values, monomeric proteins exhibited non-ideal behavior while aggregated species were not eluted. As the pH was lowered below 5, both aggregated and monomeric species were eluted, with the amount of aggregated species increasing with decrease in pH. Final elution conditions selected for the simul- taneous chromatography of monomeric and aggregated proteins were 0.1 M orthophosphoric acid, pH 2.5. The utility of the system was evaluated by determining the rates of protein degradation at elevated temperatures and comparing the results with those obtained using standard bioassay procedures. The rate of formation of aggregated species was also determined by HPSEC and corresponded to the rate of degradation of monomeric protein. The use of HPSEC with low pH eluent provides a rapid means for estimating protein stability under accelerated temperature conditions as well as for determining the existence and formation of aggregrated species.

INTRODUCTION

An important consideration in the use of recombinant DNA derived proteins for therapeutic purposes is that the protein should remain unchanged over a prolonged period of time. One means of monitoring the stability of a protein is via a specific bioassay. These, however, are often time consuming and subject to consider- able deviation. A number of other tests, including polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), may also give evidence as to the stability of the protein in question.

Over the course of investigations of some recombinant proteins, we have examined the use of high-performance size-exclusion chromatography (HPSEC) to evaluate protein stability. Size or steric exclusion chromatography is a chromatographic technique that, under ideal conditions, separates solutes on the basis of their molecular size. The elution volume of a solute is used to determine its molecular weight by comparison with the elution volumes of a series of solutes of known molecular weights. SEC, introduced in 1959 by Porath and Flodin’, has found extensive use in

0021-9673/88/%03.50 0 1988 Elsevier Science Publishers B.V.

290 E. WATSON, W. C. KENNEY

the characterization and separation of proteins. The major drawbacks of this technique have been the relatively low resolutions and long times necessary to carry out chromatographic separation of a complex sample. These limitations were widely re- alized and with the emergence of HPLC in the 197Os, the feasibility of macroporous supports for the HPSEC of proteins was also demonstrated. Currently, the most popular HPSEC suport commercially available is porous silica which has been modi- fied by reaction with hydrophilic ligands. This creates a hydrophilic layer that deac- tivates the surface on the silica support. While HPSEC packings are designed to minimize protein-stationary phase interactions, these can occur with all commercially available supports. As a consequence, true SEC is much more difficult to obtain in practice, and a number of carefully designed studies have shown that the mechanism governing the separation of proteins can be induced to deviate from a pure SEC mechanism by the composition of the eluent and by the physical condition of the column packingzp4. These interactions can be quite complex in nature and may involve ionic interactions with the silica and hydrophobic partitioning into the stationary phase.

In this report, three recombinant derived proteins, an alpha type interferon analogue (IFN-Con& gamma interferon (IFN-y), and an interleukin-2 analogue [IL-2(ala125)] were used to develop chromatographic conditions that permitted the determination of monomeric forms as well as aggregated species. Although some of these results are empirical in nature, they have important practical applications for the determination of the stability characteristics of certain proteins.

MATERIALS AND METHODS

Chromatography The liquid chromatograph consisted of a Spectra-Physics 8700 ternary gradient

pump, a Rheodyne 8 175 injector, a Shimadzu SPDdA variable-wavelength detector, and a Spectra-Physics 4270 integrator. The SEC columns, I-125 and 300SW (30 cm x 7.8 mm I.D.) were obtained from Waters Assoc. The sample injections, generally 10 ~1, at initial protein concentrations of 0.4 mg/ml, were made using Hamilton syringes. Eluents were filtered and degassed prior to use. The phosphoric acid eluents were made by weighing out the appropriate amount of orthophosphoric acid (85%) and titrating to the desired pH with 10 M sodium hydroxide. All chromatography was carried out at room temperature (ca. 18°C) and detection was at 280 nm. The excluded elution volume was determined using thyroglobulin. IFN-Coni, IFN-y, and IL-2(ala125) samples for analyses were products manufactured at Amgen.

IL-2(alal25)bioassay The bioactivity of IL-2(ala125) was determined by measuring [3H]thymidine

incorporated into an IL-2 dependent murine T-cell lines. One unit of activity is the amount of IL-2 which gives 50% of the maximum incorporation of radioactivity.

IFN-y bioassay Bioactivity of IFN-y was determined as antiviral activity in an end-point cy-

tophathic effect assay6p7.

HPSEC OF RECOMBINANT DERIVED PROTEINS 291

RESULTS

Eflect of phosphate concentration Plots of the retention time of IL-2(ala125), IFN-Con1 and IFN-), as a function

of phosphate concentration from 0.001 to 0.5 A4, pH 6.9, ‘are shown in Fig. 1. Under these conditions entirely different chromatographic behavior was observed for these three proteins.

1 I I I I t

0.1 0.2 a3 a4 a5

PHOSPHATE CONCENTRATION

Fig. 1. Retention time of IL-2(ala125) (a), IFN-y (0) and IFN-ConI (m) as a function of phosphate concentration at pH 6.9.

At very low phosphate concentrations, the retention time of IFN-Con1 almost approached the void volume value (5.80 min). As the phosphate concentration increased, there was a corresponding increase in the retention time with a plateau value being reached at about 0.1 M. The retention time remained relatively constant from 0.1 to 0.3 M phosphate but did increase at 0.5 M.

With IL-2(ala125), at phosphate concentrations less than 0.1 M, the retention time gradually increased with decreasing phosphate concentration and below 0.005 M, no IL-2(ala125) eluted from the column. The retention times were unaffected by ionic strength in the range 0.145 M.

Retention times of IFN-y were constant in the range 0.245 M, and at phosphate concentrations less than 0.2 A4, no IFN-7 eluted from the column.

pH efect Fig. 2 shows the dependence of retention times for IFN-Conr, IL-2(ala125)

and IFN-7 as a function of pH at 0.1 M phosphate. The retention times of all three proteins examined decreased with decreasing pH and in each case there was also dramatic improvement in peak resolution.

IFN-y could only be eluted when the pH had decreased to 5.0, or less. Follow- ing a series of six injections, the response increased by almost 30% by the third injection and remained essentially constant thereafter. As the pH was decreased stepwise from 5.0 to 2.5, the IFN-y peak height continued to increase.


Fig. 2. Retention time of IL-2(ala125) (a), IFN-y (0) and IFN-Con1 (w) as a function of pH at 0.1 M phosphate.

Aggregates Monomeric IL-2(ala125), IFN-Coni and IFN-), proteins were used in the

above studies to demonstrate the existence of non-ideal SEC. Since proteins are eluted in the sequence of decreasing molecular size in SEC, we were interested in determining the elution behavior of aggregated species and whether non-ideal SEC occurred to a similar extent with those species. A partially aggregated IL-2(ala125) sample was used as a test system. This sample had been previously analyzed on a Sephadex G- 75 column, and was known to contain both monomeric and aggregated IL-2 species. Moreover, it had previously been shown that the high-molecular-weight forms pos- sessed little bioactivity and were composed of monomeric units of IL-2(ala125) as evidenced by non-reducing sodium dodecylsulfate-polyacrylamide gel electrophore- sis8.

At pH 6.9 and phosphate concentrations ranging from 0.1 to 0.5 M, the only species that was eluted was monomeric IL-2(ala125) with no evidence for the presence of the aggregated species. In 0.1 M phosphate, as the pH decreased below 5, the aggregated species was eluted from the column in the void volume. The amount of aggregated species eluted continued to increase markedly with decreasing pH (Fig. 3). No studies were conducted at pH values less than 2.5 since this approached the manufacturer’s lowest recommended value for column support integrity.

2 3 4 5 6

PH

Fig. 3. Amount of aggregated IL-2(ala125) eluted as a function of pH at 0.1 M phosphate.


Rationale for use of I-125 SEC column Studies were also carried out with a 300SW column which has a molecular

weight (mol. wt.) exclusion limit of 400000. The formation of aggregates was con- sidered at that time to proceed via formation of multimeric species, and for this reason we were interested in the feasibility of determining the existence and formation rates of these various species as a function of temperature and time. No evidence was found for dimer or trimer formation and instead a broad shaped species chromatographed (results not shown). The chromatographic performance of this aggregated species made it essentially impossible to obtain quantitative information with any degree of confidence. As a result, we elected to use the I-125 column which has a mol. wt. exclusion limit of 75000. This caused all the aggregated forms to be eluted in the void volume as a sharp, discrete peak that could be readily detected and quantitated at concentrations corresponding to a few percent aggregation of monomeric IL-2(ala125) or IFN-y. This empirical observation led to consideration of situa- tions where formation of aggregate might occur and where its detection and quantitation were of potential importance.

Degradation kinetics of IL-2(ala125) and IFN-y at elevated temperatures In order to evaluate the validity of the HPSEC procedure for determining the

degradation rates of recombinant proteins, a series of studies were carried out at elevated temperatures comparing the results obtained with those using standard bioassay procedures. The degradation of IL-2(ala125) was followed at elevated temperatures of 80 and 65°C. Separate vialed samples were used throughout and the amount of IL-2(ala125) that remained was determined in each vial using both HPSEC and the bioassay procedure.

Fig. 4 shows the chromatograms obtained for IL-2(ala125) initially and after

A B

TIME (minutes)

Fig. 4. Chromatograms of IL-2(ala125) at time 0 (A), and 60 h at 80°C (B). The peak at 5.8 min is aggregated IL-2(ala125) and at 8.1 min, monomeric IL-2(aIa125).


60 h at 80°C. There is no indication for aggregation proceeding through stepwise formation of dimer, trimer, etc. In this respect, the chromatograms are relatively uncomplicated with only two species apparently present, i.e., intact monomer and total aggregate. A semi-log plot of the percentage remaining, either monomer form (HPSEC) or bioactivity, as a function of time is shown in Fig. 5. Degradation profiles determined using HPSEC, were found to exhibit apparent first order relationships (regression coefficients greater than 0.98) when the logarithmic values of residual concentrations of IL-2(ala125) monomer were plotted as a function of time. The half-lives at 80°C were 19.9 and 19.5 h, respectively, and at 65°C were 6.8 and 6.1 days, respectively, using the HPSEC and bioassay procedures.

123458 1

TIME (days)

Fig. 5. Semi-log plot of percent remaining IL-2(ala125) as a function of time at 80°C. Amount remaining analysed by HPSEC (0) and bioassay (0).

A similar study was carried out with IFN-y at 55°C. As before, only two species corresponding to monomeric and total aggregated IFN-y were evident. In general, there is excellent correlation between both procedures, with half-lives for IFN-y being 8.6 min for HPSEC and 7.4 min for the bioassay procedure, Fig. 6.

Aggregate formation at elevated temperature The next series of studies was carried out with the intention of determining the

extent and rate of formation of aggregates derived from IL-2(ala125) and IFN-1, at elevated temperatures. Fig. 7 shows the formation with time for aggregates of IFN-?/ at 55°C. The loss of monomeric IFN-JJ under these conditions is also included. The rate of formation of the aggregated fraction, determined using the method of resid- uals, was found to be 0.073 min-’ for IFN-7. The closeness of the result obtained to the degradation rate for monomeric IFN-y (0.089 min-‘), allowing for experimental error, indicates that the major route for loss of monomer for IFN-JJ proceeds through the formation of aggregated species. Similar results were obtained with IL-2(ala125) in that the rate of loss of monomer was comparable to rate of formation of aggregate (0.029 h-l vs. 0.025 h-l, respectively). There was no indication found for the formation

HPSEC OF RECOMBINANT DERIVED PROTEINS

1 10 20 30

TIME (minutes)

295

Fig. 6. Semi-log plot of percent remaining IFN-y as a function of time at 55°C. Amount remaining analyzed by HPSEC (0) and bioassay (0).

of species of mol.wt. lower than IL-2(ala125) of IFN-y, i.e., indicative of peptide bond cleavage.

Mechanical agitation Very few studies reported on the effect of mechanical stress on recombinant

proteins9. Mechanical stress has been evaluated in several ways. De Somer et al.‘O rotated samples at a fixed speed of 25 rpm. Sedmak and Grossberg” used four 30- s periods on a Vortex mixer at 4°C. Using the latter technique, but extending the conditions to five 60-s bursts at top speed, the formation of aggregates was found to

TIME (minutes)

Fig, 7. Semi-log plot for the formation of aggregated (0) IFN-1 and degradation of monomeric (0) IFN-v with time at 55°C.


be extremely low. IL-2(ala125) showed approximately 1% aggregate after 5 min of vigorous mixing and IFN-y aggregate formation was less than 1%.

Rapid freeze thaw Another area of interest was the effect of freeze-thawing on loss of monomer

and formation of aggregates. IL-2(ala125) and IFN-y were subjected to five cycles of rapid freezing in a dry-ice-propanol bath followed by thawing in water of 10°C or less. At the end of this five cycle period, the samples were analyzed for loss of initial material and presence of new components. None of these experiments indicated that loss of initial product or aggregate formation had occurred.

DISCUSSION

Silica based chromatographic supports are made suitable for HPSEC following chemical modification of the silanol groups to give hydrophilic functional groups. Ideally, for SEC purposes the surface of the chromatographic support should be only hydrophilic, but in practice the complete derivatization of the silanols is difficult to achieve. The result of this is that a percentage of silanols are present which are ionizable, Kato and Hashimoto l* have determined that the dissociation of underiv- atized silanol groups on TSK columns begins at a pH value slightly below 5. Schmidt et aL3, and Pfannkoch et aL2 have reported that all commercially available HPSEC columns that are covalently bound to silica gel contain residual silanols which exhibit anionic behavior at pH >4.

IFN-Coni consists of three species with pl values of 5.7, 6.0 and 6.1 with the 6.0 fraction representing 70% of the total. The respective pl values of IL-2(ala125) and IFN-), are 7.8 and 11. The behavior observed in Fig. 1 at low ionic strength can be explained by a consideration of the particular charge on the proteins at pH 6.9. Negatively charged proteins, here IFN-Coni, would be excluded from the negatively charged stationary phase and as a result would show decreased retention times with decreasing ionic strength. At the lower ionic strengths, positively charged IL- 2(ala125), would interact with the negatively charged silanols, and the effect would be to cause the positively charged IL-2(ala125) to be eluted with an increase in retention time. IFN-), was not eluted until the molarity was 20.2 A4 and this result is consistent with a strong ion-exchange effect between positively charged IFN-7 and the negatively charged silanol groups. At higher phosphate concentrations the elec- trostatic interactions are quenched such that little change is observed in retention times.

The increase in IFN-Con1 retention time at the highest molarity evaluated, 0.5 M phosphate, is consistent with the results reported by Schmidt et a1.3 that hydrophobic interaction of proteins with the organic bonded layer becomes the dominant interaction mechanisms at higher ionic strength.

The decrease in retention times of IL-2(ala125), IFN-y and IFN-Con, observed with decreasing pH can be similarly explained in terms of decreased ionization of the silanol groups at pH < 5. Under these pH conditions, the silanol functional groups would be protonated and would not function as cation-exchange sites.

While IL-2(ala125), .IFN-7 and IFN-Con1 could be chromatographed at 0.2 Mphosphate, pH 6.9, their aggregated species were not eluted under these conditions.


In addition, the columns began to show signs of deterioration after a period of 4-6 weeks leading to increased retention times, loss of peak symmetry and adsorption on the column. Under pH 2.5 conditions, these same columns were capable of prolonged used to such an extent that they are still operational after nine months. This indicated that a contributing factor to column deterioration observed at pH 6.9 was loss of ligand, resulting in the formation of silanol groups. The use of HPSEC with low pH conditions has since been applied to other recombinant proteins manufactured here and in all cases has allowed stability studies under extended storage and accelerated temperature conditions to be determined.

A current concern in the administration of recombinant derived proteins is the presence of aggregates in the administered samples. These can have undesirable side effects and at present there is no convenient technique for their quantitative determination. Using the HPSEC column with a low mol.wt cut-off (75 000) has the effect of causing aggregates to elute as a sharp, discrete peak and has resulted in an empirical approach for their quantitation. In this way, their existence and rate of formation can be easily followed and kinetic parameters determined. We have also extended our interests to study enzymes, such as subtilisin and analogues where aggregate formation would not be expected to occur. Under accelerated temperature conditions, no aggregates were noted and instead the generation of peptides produced by auto-digestion could be followed. These studies highlight the advantages of the HPSEC procedure to follow the complete course of protein degradation whether it be through aggregation or peptide bond cleavage.

The fact that the rate of formation of aggregates from IL-2(ala125) and from IFN-7 are essentially equal to the rate of loss of the monomers indicates that the primary route for inactivation for these proteins is through the formation of aggregated species. The apparent first-order kinetics observed in these studies suggests that the mechanism of inactivation involves transformation of the active form into a form which more readily undergoes aggregate formation. For example:

IL-2(ala125) 2 IL-2(ala125)* k2 + aggregate forms -

1

The first step is rate limiting. IL-2(ala125)* could represent a reversible de- natured form or an alternate active conformational form. Once aggregate formation occurs, e.g. dimer formation, these forms rapidly proceed to higher heterogeneous molecular weight species (2 tetramers) as evidenced by HPSEC on columns with mol.wt. exclusion limits of 70000 and 400000. Additional studies are required to define the actual mechanism involved, but the empirical observations that apparent rate constants for loss of bioactivity, loss of monomer, and formation of aggregates are essentially identical indicate that HPSEC is a powerful tool for use in stability studies. In addition, the appearance of a few percent of aggregate forms is more rapidly detected than a loss of a few percent of monomer or bioactivity thus alowing for a more rapid screening of conditions for preformulation and formulation of therapeutic proteins.

Recently, Ahern and Klibanov l3 addressed the question of enzyme inactivation using lysozyme as a model compound. In a series of studies carried out at 100°C


and at pH values of 4, 6 and 8, the results indicated that the major cause for loss of activity was due to hydrolysis of aspartic acid bonds and deamidation at asparagine residues. It was proposed that thermal stability of recombinant derived proteins would be enhanced by the replacement of asparagine. with Gln, Ile or Thr which would reduce or eliminate the rate of deamidation, while replacement of aspartic acid with Glu would minimize inactivation caused by hydrolysis of Asp-X bonds. The results found by us indicate that at elevated temperatures, IFN-7 and IL-2(ala125) are thermally inactivated largely through aggregate formation. These results suggest that a promising approach to the stabilization of some proteins may lie in the consideration of additives that would inhibit aggregate formation.

ACKNOWLEDGEMENT

IL-2(ala125) is being jointly developed by AMGEN (Thousand Oaks, CA, U.S.A.); Cilag (SchalIhausen, Switzerland); and Ortho Pharmaceutical Co. (Raritan, NJ, U.S.A.).

REFERENCES

1 J. Porath, and P. Flodin, Nature (London), 183 (1959) 1657. 2 E. Pfannkoch, K. C., Lu, F. E. Regnier and H G. Barth, J. Chromarogr. Sci., 18 (1980) 430. 3 D. E. Schmidt, Jr., R. W. Giese, D. Conron and B. L. Karger, Anal. Chem.. 52 (1980) 177. 4 H. Engelhardt and D. Mathes, Chromatographia, 14 (1981) 325. 5 S. Gillis, M. M. Ferm, W. Ou and K. A. Smith, J. Zmmunol.. 120 (1978) 2027. 6 P. K. Week, S. Apperson, L. May and N. Stebbing, J. Gen. Viral., 57 (1981) 233. 7 P. K. Week, S. Apperson, N. Stebbing, P. W. Gray, H. M. Shepard and D. V. Goeddel, Nucleic Acids

Res., 9 (1981) 6153. 8 T. Arakawa, T. Boone, J. M. Davis and W. C. Kenney, Biochemistry, 25 (1986) 8274. 9 A. M. Albisser, W. Lougheed, K. Perlman and A. Bahoric, Diabetes, 29 (1980) 241.

10 P. De Somer, M. Joniau, V. G. Edy and A. Billiau, In Vitro Monogr., 3 (1974) 39. 11 J. J. Sedmak and S. E. Grossberg, .Z. Gen. Viral., 52 (1981) 195. 12 Y. Kato and T. Hashimoto, J. High Resolut. Chromatogr. Chromatogr. Commun., 6 (1983) 45. 13 T. J. Ahern and A. M. Klibanov, Science (Washington, D.C.), 228 (1985) 1280.

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High-performance size-exclusion chromatography of recombinant derived proteins and aggregated species