Anal. Chem. 1996,67, 447-455
Affinity Purification and Microcharacterization of Recombinant DNA-Derived Human Growth Hormone Bsolated from an in Vivo Model John E. Batternby,* Venkat R. Mukku, Ross G. Clark, and William S. Hancockt Genentech, Inc., South San Francisco, California 94080
A procedure has been developed for the isolation and p d c a t i o n of trace amounts of unlabeled proteins from biological solutions. Using a combination of aftinity chromatography and reversed-phase HPLC, microgram amounts of recombinant DNA-derived human growth hormone (rhGH) were p d e d from an in vivo rat model. Microcharacterization techniques were developed, and picomole amounts of the recovered protein were digested with trypsin and characterized using capillary HPLC peptide mapping. The described procedures were used to study the chemical changes that occur in rhGH following intravenous administration. The study demonstrated that both deamidation and oxidation can occur in vivo, although the former would occur to a si@cant extent only in proteins with an extended half-life. Metabolism studies using protein pharmaceuticals are a challenging exercise for the analytical chemist, with low sample concentrations, complex matrices such as plasma, and the complexity of the macromolecule as primary difficulties.' In the past, radioactive labels have been used to follow the fate of proteins. The label was introduced into the protein by techniques such as radio iodination of tyrosine residues or by labeling of amino groups by acylation reactionse2 The labeled protein can then be used to measure important factors such as pharmacokinetics, tissue distribution, and metabolism. In metabolism studies, electrophoretic methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are used to follow proteolytic events as well as the appearance of higher molecular weight species. Nondenaturing PAGE conditions or sizeexclusion chromatography are used to follow higher molecular weight species formed either due to aggregation of the protein or by interaction with binding proteins. The detection of the protein can be achieved by either autoradiography or antibody-based approaches.lS3 However, these analytical techniques cannot readily detect degradative reactions such as deamidation, isoaspartyl formation, methionyl oxidation, and minor proteolytic events. Additionally, the use of radioactive labels has other disadvantages: many studies in humans are not possible, the introduction of the label may perturb the metabolic process as well as the chromatographic properties of the protein, and the loss of the label + Resent address: Hewlett-Packard Co., Palo Alto, CA 94304. (1) Ferraiolo, B. L.; Moler, M. In Protein Pharmacokinetics and Metabolism; Ferraiolo, B. L.,Moler, M., Gloff, C A , Eds.; Plenum: New York, 1992. (2) Bolton, A E.; Hunter, W. M. Biochem. J. 1973,133, 529-538. (3)Janis, L. J.; Regnier, F. E. Anal. Chem. 1989,61, 1901-1906.
0003-2700/95/0367-0447$9.00/0 0 1995 American Chemical Society
from the protein, particularly with the use of iodine as a label, is problematic. In the last few years, analytical protein techniques have become much more powerful with the advent of capillary HPLC, highperformance capillary zone electrophoresis (CE), and advances in mass spectrometry (MS) such as electrospray ionization and laser des0rption.4,~The application of these techniques can allow the analysis of subtle chemical changes occurring in polypeptide samples at the low picomole to even the low femtomole level. With such a high degree of sensitivity, it may be possible to monitor degradative reactions that occur in vivo. Such approaches cannot be applied directly to biological samples due to the low levels of individual proteins and the complexity of a medium such as serum. In these cases, concentration and pudication steps are necessary. The design of these steps is important because small amounts of proteins are readily lost, e.g., by adsorption to container walls. Such losses are generally more serious at the later stages of purification, when the partially purified protein is no longer protected by higher concentrations of other serum proteins. Recently Regnier et al. published a procedure that coupled afflnity chromatography with HPLC for the capture of proteins from biological matrices? We decided to use a variation of this approach in which the capture step was based on a binding protein that had been shown to correspond to the soluble extracellular domain of the growth hormone receptor.'j The binding protein was used rather than an antibody because it had been previously established that it was capable of binding rhGH variants, e.g., deamidated, cleaved, aggregated, and oxidized, produced by degradation reactions. This report will describe the use of a receptor protein immobilized on agarose, coupled with reversedphase HPLC (RP-HPLC) to isolate small amounts of recombinant DNA-derived human growth hormone (rhGH) from serum and tissue culture samples. This approach was further extended by the development of small-scale trypsin digestion procedures that allowed capillary HPLC mapping of the peptide fragments with either W or electrospray mass spectrometric detection. EXPERIMENTAL SECTION
Samples and Reagents. rhGH is a product of Genentech Inc., and rhGH binding protein (rhGHbp) was purified from a (4) Wiktorowicz, J. E.; Colburn, J. C. In Capillary Electrophoresis. nieoty and Practice; Grossman, P. D., Colbum, J. C., Eds.; Academic Ress, Inc.: San Diego, CA, 1992. (5) Cam, S. A; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802-2824. (6)Fuh, G.; Mulkerrin, M. G.; Bass, S.; McFarland, N.; Brochier, M.; Bourell, J. H.; Idght, D. R; Wells, J. A]. Biol. Chem. 1990,265, 3111-3115.
Analytical Chemistry, Vol. 67,No. 2,January 15, 1995 447
mammalian cell line by P. Mckay, Genentech Inc.'j TPCK-treated trypsin was purchased from Worthington Biochemical Corp. Tween 20 was purchased from Sigma Chemical Co. Trinuoroacetic acid W A ) ,HPLC/spectro grade, was obtained from Pierce Chemical Co. Acetonitrile (HPLC grade) was purchased from Burdick and Jackson. Milli Q water was produced by a Millipore water purification system. CNBr-activated Sepharose 4B was purchased from Pharmacia. rhGHbp Immobilization. The rhGHbp (1.0 mg/0.5 mL) was exchanged into coupling buffer (0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3) using a NAP5 column (Pharmacia), CNBr-activated Sepharose 4B (0.5 g) was swollen in 1 mM HCl(5 mL) for 15 min and then washed with aliquots of the same solution (100 mL). The gel was washed with coupling buffer (5 mL) , immediately transferred to rhGHbp (1mg) in coupling buffer (2 mL), and incubated for 2 h at room temperature. The reaction mixture was filtered, and the content of rhGHbp in the filtrate was determined by OD 280 nm measurement. To the filtered gel was added 0.2 M glycine, pH 8.0 (1mL), and this was allowed to react for 2 h at room temperature to block remaining active groups. Noncovalently bound rhGHbp was removed by multiple washings (5 times each), alternating between coupling buffer (0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3) and acetate buffer (0.1 M sodium acetate, 0.5 M sodium chloride, pH 4.0) and then with elution buffers (0.1 M citrate, pH 3.5; 0.2 M glycine, pH 2.4; 3 M potassium thiocyanate in PBS, pH 7.4). A similar procedure was used for coupling antibodies to the CNBr-activated Sepharose 4B. Stability of rhGH in Rat Serum. Samples of rhGH (10 pg) were added to either rat serum (1 mL) or PBS (1 mL). Sodium azide was added to the samples to 0.02% (10 pL of 2% sodium azide), and the samples were incubated at 37 "C. At time zero and then every 24 h, a serum sample and a PBS sample (control) were removed and stored at -70 "C until each was purified on an immobilized monoclonal antibody column. Incubation of rhGH with a Mouse Myeloleukemic Cell Line. FDGPl is a mouse myeloleukemic cell line. These cells do not have growth hormone (GH) receptors and therefore do not respond to human GH (hGH). Upon transfection with the hGH receptor gene, these cells proliferatein the presence of hGH.7 These latter cells were routinely cultured in RPMI medium containing 10%fetal bovine serum, 1pg of mercaptoethanol, 400 pg/mL Geneticin (G418), and 2 mM glutamine in a 37 "C incubator with 5%carbon dioxide and 100%humidity. For in vitro metabolism studies with rhGH, a cell suspension of lo6 cells per milliliter were incubated in the above medium, but with 5%fetal bovine serum, in the presence of 100 ng/mL rhGH for 24 h. Medium containing 100 ng/mL rhGH, but without cells, served as a control. The cell suspensions were centrifuged at 500g for 10 min. The supernatants were further centrifuged at lOOOOg to remove any cell debris and stored frozen at -70 "C until analysis. Preparation of in Vivo Metabolism Samples. Four adult rats (female dw/dw, Simonsen Laboratories, Gilroy, CA) were each given a 4 mg (1 mL) intravenous dose of rhGH, and terminal bleeds were performed at 5,15, and 45 min and at 1h. Separated serum was stored at -70 "C until it was purified. Affinity Chromatography. The affinity matrix (CNBractivated Sepharose 4B with immobilized rhGHbp or antibody) was packed into a column (Pharmacia, HR 5/2) to a bed height ~
(7') Colosi, P.; Wong, K; Leong, S. R J. Bid. Chem. 1993,268, 12617-12623.
440 Analytical Chemistry, Vol. 67, No. 2,January 15, 1995
of 5 mm and preconditioned with bovine serum albumin @SA) diluted in PBS (40 mg/5mL). Rat serum (0.5 mL) obtained from terminal bleeds or other rhGHcontaining samples was diluted with PBS containing BSA, 2 mg/mL (4.5 mL), and loaded over 90 min. For recovery calculations,the initial serum rhGH content was determined using an enzyme-linked immunosorbent assay (ELISA), and the yield of captured rhGH was determined by peak areas obtained from the secondary RF-HPLC purification and quantitation step (see below). Similarly, the flow-through was collected and measured for the content of nonbound rhGH. Nonspecifically bound proteins were removed by washing with PBS containing 1 M sodium chloride and 0.05%polysorbate 20, pH 7.4 (15 mL), followed by PBS (5 mL). Elution of specifically bound rhGH was carried out using 0.2 M glycine-HC1, pH 2.4 (5 mL). The afsnity column was regenerated by washing with the following (10 mL each): potassium thiocyanate/PBS (3 M), PBS, alternating coupling buffer and acetate buffer, (five times each), and PBS. When not in use, the affinity column was stored at 0-4 "C in 0.05% Thimerisal diluted with PBS. Secondary Rp-HPLC Purification and Quantitation. The eluate from the af6nity column was further purified, concentrated, and quantitated using RP-HPLC on a Vydac RPC4 column (4.6 mm x 25 cm, 300 k pore, 5 pm particle). A manual injector (Rheodyne) was fitted with a 1 mL sample loop, and the total eluate from the affinity column was loaded by multiple injections. Solvent A was 0.1% TFA, and solvent B was 0.09% TFA in acetonitrile. The column was equilibrated in 20%solvent B. After the column was held at 20%solvent B for 7 min, a rapid gradient was run to 60%solvent B over 18 min. The flow rate was 1mL/ min, and the absorbance was monitored at 214 nm. Samples were collected in siliconized Eppendorf tubes and stored at -70 "C until they were needed for characterization. Quantitation was determined by comparison of peak areas with those obtained for known amounts of standards analyzed under the same conditions. Determination of rhGH Variant AEdties. To determine if the aftinity purification step recovered rhGH variants with equal affinities, a mixture consisting of 2 pg each of twochain rhGH, des-Phe1Pro2-rhGH,deamidated rhGH, dimeric rhGH, and rhGH in PBS (5 mL) and containing 50 mg of BSA was loaded onto the afsnity column and purified as described above. The single peak, consisting of rhGH and variants, collected from the secondary RP-HPLC purification and quantitation step, was then reanalyzed using different RP-HPLC conditions (PW-S column, 300 k pore, 8 pm particle, 4.6 mm x 15 cm column) that now resolved these variants, and the results were compared to a similar analysis of the starting mixture. Solvent A was 50 mM potassium phosphate, pH 7.5. Solvent B was acetonitrile. The column was equilibrated with 20% solvent B. The sample was loaded and held at these initial conditions for 10 min, and then a gradient to 44% solvent B was run over 5 min, and the sample was held for a further 30 min. A final gradient to 60%solvent B was run over 5 min to complete elution. The flow rate was 0.5 mL/min, and the absorbance was monitored at 214 nm. Standard Trypsin -stion and Peptide Mapping. Samples were exchanged into trypsin digest buffer (100 mM sodium acetate, 10 mM Tris base, and 1 mM calcium chloride, pH 8.3), using NAP5 columns (Pharmacia). Alternatively,smaller volume samples (less than 20 pL) were simply diluted with two volumes of digest buffer, provided no change in the pH occurred. Samples were digested by addition of VCK-treated trypsin (Worthington
Biochemical) dissolved in 1 mM HCl (1:100trypsin:protein w/w) and incubated at 37 "C for 2 h, followed by a second enzyme addition to give a h a l concentration of 1:50 (trypshprotein w/w). At the end of the digest (4 h), the pH was lowered to 2-3 with 5%WA All digests were carried out on the nonreduced protein and resulted in two disuhide-linked peptides being present in the digest mixture (TGT16 and T2@T21). RP-HPLC tryptic mapping was performed using a Nucleosil C18, 5 pm particle, 300 A pore size, 2.1 mm x 15 cm column. Solvent A was 0.1% TFA, and solvent B was 0.1% TFA in acetonitrile. Elution involved a 5 min isocratic hold at 100%solvent A, followed by a linear gradient (0.3%/min) to 40%solvent B. A 4%/min linear gradient to 60% solvent B with a 10 min hold completed elution. The column was returned to the initial conditions over 5 min and reequilibrated with 100%solvent A for 25 min. The flow rate was 200 pL/min. Temperature was 40 "C, and W absorption was monitored at 214 nm. Recoveries were based on peak areas (f2% except for hydrophobic peptides, which can give greater variability under conditions that promote nonspecific interactions8-lI). The detection limit for a variant peptide was 5%,and the amount was determined from the absorbance at 214 nm relative to the absorbance of the unmodified peptide.1°
Small-ScaleTrypsin Digestion and Capillary HPLC Peptide Mapping. Typically a small amount of rhGH (0.1-5 pg), either dried or near dryness, in an Eppendorf tube, was diluted with digest buffer (2 pL). Trypsin (1:20 w/w protein:enzyme (P: E)) dissolved in digest buffer (1 pL) was added. The digest mixture was incubated at 37 "C for 2 h, followed by a second enzyme addition (1:lO w/w PE). The digest was left at room temperature overnight and then acidified with 2%TFA (3 pL). In studies to improve recoveries of tryptic fragments, the digested samples were made to 0.15%Tween 20 before the LC map was run. The capillary LC system has been described previously.* Using this system, the released tryptic fragments were resolved on a Nucleosil C18,3 pm particle, capillary column (320 pm x 15 cm). Solvent A was 0.1%TFA, and solvent B was 0.08%TFA in acetonitrile. The total volume of the digest mixture (or the portion that contained less than 1 pg) was loaded onto the capillary column equilibrated in 100%solvent A Separation of the tryptic fragments was achieved by running a 0.3%/mingradient to 40% solvent B, followed by a 2%/min gradient to 60%solvent B. The flow rate was 3.5 pL/min, and detection of the released peptides was by UV absorbance at 214 nm or by total ion current (TIC) measured by electrospray mass spectrometty. RESULTS M t y Chromatography. The eluate from the affinity
column was not monitored directly due to large absorbance (8)Battersby, J. E.; Guzzetta, A. W.; Hancock, W. S. The Application of Capillary LC to Biotechnology,with Reference to the Analysis of Recombinant DNADerived Human Growth Hormone (rhGH). /. Chromatogr., in press. (9)Chloupek, R C.; Battersby,J. E.; Hancock, W. S. In HPLC ofpeptides and Proteins: Separation Analysis and Conformation; Mant, C. T.,Hodges, R S., Eds.; CRC Press: Boca Raton, FL, 1991. (10)Bennett, W.F.;Chloupek, R;Harris,R;Canow-Davis, E.; Keck,R;Chakel, J.; Hancock, W. S.; Gellefors, R;Paulu, B. In Advances in Growth Hormone and Growth Factor Research; F'ythagora Press: Rome, 1989; pp 29-50. (11) Hancock, W.S.; Canova-Davis, E.; Chloupek, R C.;Wu, S. I.; Baldonado, I. P.; Battersby,J. E.; Spellman, M. W.; Basa, L. J.; Chakel,J . AIn ?%erapeutic Peptides and Proteins: Assessing the New Technologies; Banbury Report 29; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1988,pp 95-117.
1.
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.
,
10
.
. Time
.
.
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.
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Figure 1. Secondary RP-HPLC purification step performed on eluate from an affinity capture of rhGH (upper profile). The sample was loaded, using multiple 1 mL injections, onto a Vydac C4 column and eluted with acetonitrile. The lower profile is a similar analysis of a mixture of rhGH and variants. The separation conditions are described in the Experimental Section.
changes relative to the small sample load that occurred as a result of changing buffers. To optimize the affinity purification,we used ELISAs to measure the amount of binding of the target protein at each step of the purification scheme. These values were in agreement with the yield of captured rhGH estimated by peak areas from the secondary W-HPLC analysis. Using the procedures described in the Experimental Section, the reproducibility of the affinity chromatographybinding was excellent (with a range of 7040%)and was not dependent on the loading conditions. To measure purity we developed a RP-HPLC assay (see secondary W-HPLC purification and quantitation) which could provide both quantitative and purity information in the same step. Using native rHGH and the W-HPLC assay to guide develop ment of the affinity step, we incorporated the use of a detergent (Tween 20) in a wash step that was designed to remove nonspecifically adsorbed proteins. This improved wash, however, was also responsible for the removal of 10-15% of our target molecule (rhGH), but this was an acceptable loss in our search for a rapid, high-purity and high-yield capture step. It was not determined if the 10-15% removed was specifically or nonspecifically adsorbed. Secondary RP-HPLC Purificationand Quantitation. The eluate from the af6nity capture step, containing the partially purified protein, typically in a volume of 2-4 mL, was loaded onto a W-HPLC column using multiple 1 mL loads with a Rheodyne injector. The initial conditions (20%solvent B) were chosen so that all the rhGH variants were retained by the column. The initial mobile phase conditions were held for approximately 1.25 min/ mL of sample loaded. The rapid gradient following these initial conditions was designed to resolve rhGH species from other serum proteins and yet elute rhGH and any variants essentially as a single sharp peak (Figure 1, lower profile, retention time 26.5 miin). This peak was collected and either frozen or dried (in a speed vacuum) and then stored at -70 "C. The small peak eluting at 21 min has a retention time the same as that of BSA rhGH was the most hydrophobic species observed and consequently eluted last in the gradient and was well resolved from other serum proteins. The absence of significant levels (less than 5%) of species not related to rhGH coeluting in this sharp peak was confirmed by tryptic mapping. Determination of rhGH Variant Selectivities. A sample of rhGH and four variants (2 pg each in 5 mL of PBS) was isolated Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
449
1
4
30
40 Time (man.)
50
60
Figure 2. RP-HPLC separation of a mixture of rhGH and variants (lower profile). The upper profile is a similar separation but after isolation using the purification scheme described in this manuscript. The separation was performed on a PLRP-S column using phosphate buffer, pH 7.5 and an acetonitrile gradient. Peak 1, two-chain rhGH; peak 2, des-PhelPro*-rhGH; peak 3, coelution of deamidated and authentic rhGH; peak 4, dimeric rhGH. Details of the separation conditions are described in the Experimental Section.
and purilied from BSA using the scheme described earlier. The variants were two-chain rhGH, des-PhelProz-rhGH,deamidated material, and dimeric rhGH. The twochain variant is formed during the manufacturing process by a single proteolytic clip between tyrosine 142 and threonine 143, with the two chains being connected by an existing intrachain disulfide bond between cysteine residues 53 and 165. In contrast to the proteolytic process that forms two-chain rhGH, a chemical degradation results in the loss of two residues, phenylalanine and proline, from the N-terminus to form the des-PhelProz-rhGHvariant. Deamidated rhGH is the result of deamidation of asparagine, residue 149, and forms an aspartic acid residue, predominantly linked in the ,8 isomer form. The fourth variant in the mixture was a covalently linked dimer of rhGH. Following the afhity capture of a mixture of these variants, a single peak was collected from the RF-HPLC step and then reanalyzed using different RP-HPLC conditions to resolve these variants (see Experimental Section, Determination of rhGH Variant Affinities, for chromatographic conditions), and the result were compared to a similar analysis of the starting mixture. Baseline resolution was achieved for all components except between deamidated rhGH and rhGH, retention time 3539 min (Figure 2), and approximately 70%recovery was achieved. These components were recovered in a ratio similar to that of the starting mixture (lower profile), indicating similar recoveries of each component at this concentration. Therefore, if these variants were to be formed as the result of metabolic events, then this purilication scheme can capture these variants with good recoveries and without distorting their relative amounts. Since all the tested variants were captured, we were hopeful that other untested variants, resulting from an early metabolic event, would also be captured in a like manner. Microdigestion and Capillary LC Peptide Mapping. In 2 pL of digest buffer was digested 0.2 pg (8.7 pmol) of rhGH, with trypsin (1:lO w/w), and the resulting peptide map performed on a capillary (320pm id.) Nucleosil C18 column is shown in Figure 3, bottom profile. The gradient used in this separation was from 100%solvent A to 40%solvent B in 1 h at a flow rate of 3.5 pL/ min. The elution order of the peptides, identified using mass spectrometry, was the same in capillary (Figures 3, 6, and 7) and narrow bore (Figures 4 and 5) peptide maps and was not affected 450 Analytical Chemistry, Vol. 67, No. 2, January 75,7995
, 20
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40 50 Time (min)
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70
Figure 3. Improved recoveries in the capillary HPLC peptide maps of rhGH as a result of the addition of detergent. The lower profile is from the digestion of 0.2 p g of rhGH, and the upper profile is a repeat analysis with the addition of 0.15% Tween 20 to the sample. The peptides separated with low recoveries are marked with arrows. The peptides were separated on a Nucleosil C18 capillary column (320 pm i.d. x 15 cm) using an acetonitrile gradient (0-40% in 1 h) with a flow rate of 3.5 pUmin. Details are given in the Experimental Section.
by the gradient slope. Recoveries of peptides from the capillary HPLC peptide maps were not determined directly; however, these were estimated by comparing relative peak areas to those obtained for larger scale peptide maps whose recoveries had been previously determined to be 98%, except for hydrophobic peptides, which gave lower and more variable recoveries?-ll Low recoveries of the later eluting peptides (Figure 3, later than approximately 59 min; all other maps, later than approximately 95 min) are observed and are attributed to nonspecilic adsorption. These involved tryptic fragments T4, T10, T6-Tl6, and T9. The last two of these peptides are known to give recovery problems even under some standard analytical conditions? The use of nonionic detergents can significantly improve the recoveries of these peptides, and this is demonstrated in the upper profile of Figure 3, which was produced using the same sample as used for the lower analysis but with the addition of detergent (0.15%Tween 20). Stability in Rat Serum. The stability of rhGH in rat serum was studied, and the tryptic maps (narrow bore, 2.1 mm i.d.) used to follow any potential changes are shown in Figure 4. The digests were performed on approximately 8 pg of material. The most significant changes observed in this study were the small decrease in tryptic fragment T15 (63.5 min) and the appearance of a new peak at 64 min (see arrow, top profile, Figure 4A and B). This new peak is the result of deamidation of asparagine, residue 149, and was confirmed by mass spectrometry as described in ref 8. This occurred for rhGH incubated in either serum or PBS and increased with increasing time of incubation. rhGH recovered from rat serum at time zero (Figure 4A and B, lower profile) contained 6%of this new peak, whereas by day 3 it had increased to 21% (Figure 4A and B, upper profile). A similar increase to 17%was observed when rhGH was incubated for the same time but in PBS alone (Figure 4A and B, middle profile). For
I.,
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60 Time ( m l n . I
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65
20
40
60
' 8 0 Time (min)
120
,
Figure 6. Capillary HPLC peptide map of rhGH recovered from rat serum taken 5 min after an intravenous dose of rhGH (upper profile). The sample was recovered using the purification scheme described in the text. The lower profile is the peptide map of native rhGH. The peptides were separated on a Nucleosil C18 capillary column (320 pm i.d. x 15 cm) using an acetonitrile gradient (0-40% in 2 h) with a flow rate of 3.5 pUmin. The separation details are described in the Experimental Section.
70
Figure 4. (A, top) Peptide maps (narrow bore) of rhGH spiked into rat serum and recovered either immediately (lower profile) or after incubation at 37 "C for 3 days (upper profile). The middle profile is a similar map of rhGH recovered after spiking into PBS and incubation at 37 "C for 3 days. All three samples were recovered using the purification scheme described in the text. Deamidated T15 is shown with an arrow. (A similar peptide map of native rhGH is shown in Figure 5,top profile.) The peptides were separated on a Nucleosil C18 column (2.1 mm i.d. x 15 cm) using an acetonitrile gradient, and details are given in the Experimental Section. (B, bottom) Enlargement of the figure above between 45 and 75 min.
/ ET 0
" 100 .
1
i io
do
io
do
io
160 Time (min)
iio
ilo
150
Figure 7. Capillary HPLC peptide maps of rhGH recovered from rat serum taken 15 (middle profile) and 45 min (upper profile) after an intravenous dose of rhGH. The lower profile is the tryptic map of native rhGH. Arrows link methionine-containing peptides with their corresponding oxidized forms. The separation conditions are described in Figure 6.
r 1 7 w1. . _
20
40
60 Time
80 (mln.)
100
128
140
Figure 5. Narrow bore peptide maps of rhGH recovered from cell media after incubation either with cells (lower profile) or without cells .(middle). The arrows in the lower profile indicate regions of change. Both samples were recovered using the purification scheme described in the text. The top profile is the peptide map of native rhGH. The peptides were separated as described in Figure 4.
comparison purposes, native rhGH contained 3%of this deamidated variant (Figure 5, upper profile). Two other small peaks (39 and 56 min) are observed in the maps of both samples purified from serum (Figure 4A, upper and lower profiles) but are not seen in the map of the non-serumcontaining sample (Figure 44, middle profile) nor in the map of native rhGH (Figure 5, upper profile). This would tend to suggest
the copurification of trace amounts of serum proteins or the presence of very low levels of metabolites. The small differences observed in the peptide maps at 96,102, 106, and 111.5 min of Figure 4A are due to chymotryptic-like cleavages caused by minor differences in the digest conditions and the length of time between completion of the digest and the running of the maps. The time difference between the first and fourth analysis on any one day is more than 10 h, and although the digest has been stopped, some minor changes continue to Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
451
occur! Other significant changes are the previously observed, variable recoveries of the later eluting peptides (Figure 4A, fragmentsT6-T16,108.5 min, and T9,136 min) and the broadening of T14 (23 min), which are, therefore, presumed to be method related rather than due to sample ~ariation.~ Isolation of rhGH from Cell Culture Media. Two separate purifications were performed on samples containing rhGH incubated at a concentration of 100 ng/mL in cell media. One sample had been incubated with murine myeloleukemic cells (106/mL), and the other had been incubated in the absence of these cells. The loading of the 100 mL sample (after removal of cells were appropriate) onto the affinity column took approximately 6 h. The flow-through was collected on ice and stored at -70 "C for further studies. The affinity column was eluted (without on-line monitoring) using 5 mL of 0.2 M glycine-HC1. The collected eluate was then loaded onto the secondary RP-HPLC purification column, and the UV profiles obtained were very similar to those shown in Figure 1. The main peak was collected between 26.1 and 27.0 min, and from peak areas, recoveries were determined to be 89% when the sample was incubated with cells and 73% when the sample was incubated without cells. These recoveries are typical of previous af6nity purifications (range 70-90%) from either serum or from PBS alone, and the differences in recoveries are due to the variability of the method. The tryptic maps of these two samples performed as described in the Experimental Section, Standard Trypsin Digestion and Peptide Mapping, are shown in Figure 5. Approximately 7 pg of each sample was used for the peptide maps. The lower profile is of the sample incubated with cells. The middle profile is of the sample incubated without cells, and for comparison purposes the upper profile is the tryptic map of native rhGH. Only minor differences are observed in the map of rhGH incubated in the presence of cells (lower profile), and these are marked with arrows. The last three of these arrows indicate late eluting peptides with poor recoveries. Contamination of the purified samples with proteins other than rhGH would result in multiple small peaks in the map, and the absence of such peaks indicates the high purity achieved in this purification. In Vivo Results. Rat blood samples taken at 5, 15, and 45 min and 1 h after being dosed with rhGH were purified as described earlier. The single peak collected from the secondary RP-HPLC purification step had peak areas equivalent to 38, 4.2, 1.2, and 1.3pg of rhGH, respectively. Recoveries from the affinity column for the 5 min and 1 h samples were 58% and 71%, respectively. These values were calculated using initial rhGH serum concentrationsas determined by ELISA Recoveries were not calculated for the 15 and 45 min time points. Recovered rhGH from the 5 min sample was submitted for N-terminal sequence analysis, and the results were consistent with the known sequence of rhGH. No minor sequences were found (data not shown). This eliminates the possible action of a unknown protease, with the same specificity as trypsin, having acted on the protein and gone undetected in the tryptic map. Approximately 18pg of rhGH recovered from the 5 min sample was digested with trypsin, and the capillary tryptic map is shown in Figure 6, upper profile. For comparison purposes, the map of native rhGH is shown (lower profile). The relatively large amount of substrate (18 pg) meant that conventional digest conditions could be used, and the near maximal load (0.9 pg) injected onto the capillary column ensured good recoveries of the later eluting 452
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peptides (TlO, 103 min; T6-T16,108 min; and T9,131 rnin). The tryptic map of the 5 min sample is very similar to that of the native material, with the only change being the appearance of a new peak (66 min) having the same retention time as expected for oxidized T2 (Figure 6). This was confirmed as oxidized T2 by electrospray mass spectrometry, with the observed mass being 16 amu greater than that of T2 (data not shown). From integrated peak areas, this oxidized T2 fragment is approximately 9%of the total T2 content. No other significant changes attributable to metabolism were observed in the peptide map (see earlier discussion of nonspecific cleavages). Comparison of the partial peptide maps for rhGH recovered from the 15 and 45 min samples (Figure 7) shows increasing amounts of oxidized T2 (66 min) and decreasing T2 content (76 min). Oxidized T2 had increased to 74%of the total T2 content by 45 min. These maps were run on a different capillary column than that used to produce the map for the 5 min sample and resulted in the coelution of tryptic fragments T18-Tl9 and T1 (79 min). These maps show several other trends over time, and all involve methionine-containing peptides: the decrease in peak T11 (97 min, containing methionine at residue 125) and the increase in the corresponding oxidized peptide, retention time 81 min; the decrease in the peak with retention time 74 min, i.e., T17-Tl8-Tl9, containing methionine at residue 169, and the increase in the corresponding oxidized peptide, retention time 68.5 min (this is not easily observed due to the coelution of T8); the decrease in peak T18-Tl9 (79 min, containing methionine at residue 169 and also coeluting with T1) and the increase in the corresponding oxidized peptide (71 min). All of these assignments were confirmed by on-line electrospray mass spectrometry.8 DISCUSSION An emerging frontier in analytical biotechnology is the devel-
opment of suitable systems for observing the metabolic fate of protein pharmaceuticals. Such an understanding may speed up the drug development process by allowing better selection of candidate molecules. Currently, the chemical complexity of proteins often inhibits the detailed chemical analysis of samples from pharmacokinetic and metabolism studies compared to the relative ease of characterization of small organic molecules. In addition, as the biotechnology industry matures, the types of molecules that are being developed for example, humanized antibodies, are much more complex than those known in the early days of the industry, and methods to determine their in vivo fate need to be developed. In the introduction we outlined an approach to metabolism, based on recent advances in separation science, that has the potential to generate much more detailed chemical information about the metabolic fate of unlabeled proteins. It is important to distinguish between early and late metabolic events, especially if a key strategy was to use an initial capture step based on biorecognition. Thus, a study of early metabolic events would use an antibody or a receptor protein that has broad specificity for a molecule (metabolite) that has many of the surviving epitopes of the starting molecule. Conversely, if one wanted to study a later metabolic event, for example, intracellular processing by cathepsins, one could use an antibody directed against the major fragments known to be produced. The more significant the metabolic event(s), the greater the uncertainty that the modified molecule will not have retained sufficient intact epitopes to be
successfully recognized and captured. In this situation, one may need more than one capture step. Choice of rhGH as an Initial Study System. We chose initially to study the metabolism of growth hormone for the following reasons. We had extensive experience in the highresolution LC separations and characterization of degradation products of rhGH.loJ1Also, a number of reagents were available for the development of an efficient capture step, e.g., mono- and polyclonal antibodies to rhGH and a soluble fragment of the extracellular domain of the growth hormone receptor, in which the transmembrane and intracellular portions had been deleted, leaving the so-called growth hormone binding protein, rhGHbp. There is also an extensive body of literature on in vivo metabolism of growth hormone and the possible production of active variants.12 The characterization of growth hormone fragments isolated from a metabolism study may give clues to the existence of such variant forms and aid the process of drug design. The study of growth hormone is a realistic model for many metabolism studies since it is present at low levels in circulatory systems, e.g., in man, at 0-10 ng/mL of blood. Alternatively, if an animal is dosed with a larger than clinically relevant amount of hormone, then samples collected at later time points will still involve the characterization of low levels of material due to the rapid clearance of this protein. This rapid clearance has been demonstrated in rats using labeled material, which has shown that within 1 h, a 4 mg intravenous injection of growth hormone will be cleared to microgram 1e~els.I~ In this study we used a relatively high dose of rhGH to overwhelm any rat growth hormone binding protein present in vivo and to ensure that free rhGH was available for capture. The rat growth hormone binding protein is capable of binding human growth hormone, but the opposite is not true, i.e., human growth hormone binding protein does not bind rat growth hormone; therefore, endogenous levels of rat growth hormone were not a concern. Future studies that are directed at examining the metabolism of growth hormone at or near physiological concentrations must address this issue of capture of the growth hormone-receptor complex by use of antibodies that can bind this complex. Design and Validation of Capture Step. M e r some initial exploratory studies, we decided to use the growth hormone binding protein instead of monoclonal or polyclonal antibodies in the affinity step. The binding protein had been characterizedand shown to bind with high affinity to a variety of growth hormone variants (unpublished results). Also, by using the immobilized binding protein instead of an antibody for capture, we could detect metabolites capable of interacting with cell receptors and thus aid in a better understanding of pharmacodynamics. A key part of our strategy was the coupling of a RF-HPLC step to the initial aftinity chromatography isolation. The reversed-phase column achieves several important operations simultaneously. The captured protein can be further purified, it is concentrated and desalted, and the quantitation allows the optimization of the a€iinity chromatography step. It was also necessary to develop a similar separation, but in the capillary format, so that the samples from metabolism studies, available only in submicrogram amounts, could be detected with sufficient sensitivity. ~
~
~~
(12)Baumann,G. Endocrinol. Reo. 1991,12, 424-429. (13) Mordenti, J.; Chen, S. A; Moore, J. A; Ferraiolo, B. L.;Green, J. D. Pham. Res. 1991,8,1351-1359.
With this approach it was possible to capture in high yield (range of 70-90%) a few micrograms of metabolites in the presence of many tens of milligrams of serum proteins. The high levels of serum proteins, e.g., serum albumin, helped to reduce nonspecific losses of growth hormone and its variants. All new columns were preexposed to serum proteins to minimize nonspecific losses. The small losses that were observed were attributed to traces of material in the flow-through fractions (as measured by ELISA) and nonspecific binding. It is probably unrealistic to expect 100%recovery of such small samples, and we feel that the recovery observed in this study was sufficiently high to allow the observation of significant metabolic events. The conditions of the reversed-phaseseparation were chosen to elute the metabolites as a single peak so that quantitation of the capture step could be performed. Nonspecific binding can greatly reduce the degree of purification achieved in affinity chromatography due to interactions with the chromatographic support, the spacer arm, or the affinity ligand.14 A key step in this affinity procedure was a wash step (PBS/l M NaCl, 0.05%Tween 20) designed to reduce nonspecific binding. The binding of growth hormone and growth hormone variants to the affinity ligand was Sufficiently strong to allow the use of this detergent. In other studies, such as with tissue plasminogen activator/antibody column, such a wash step resulted in the stripping of the bound protein (unpublished observation). The careful optimization of the affinity chromatography step resulted in a 500Cbfold purification. Despite this high degree of purification, the W-HPLC analysis indicated that there were still trace amounts of serum proteins, such as albumin, in the sample, and these were therefore removed. Common growth hormone variants were recovered with a yield similar (70-90%) to that of rhGH. This observation allowed the authors to have confidence that this system could be used to study early metabolic events in vivo, but future experiments are required before we understand the limits of this system and its ability to detect novel degradation products and later metabolic events. Another challenge was to reduce the scale of the trypsin digestion, which had typically used 50-100 pg of substrate in a volume of 100 pL and with 1%(w/w) trypsin added.1° Novotny has shown that it is possible to cany out microdigestions of proteins, and we have used a modification of his conditions in this study.15 We have found, however, that slight changes in the digest conditions can result in significant changes in the map. For example, when the substrate concentration is reduced from 2 to 0.1 pg and all other parameters are kept the same, fragment T10 undergoes secondary cleavages to different extents. Such changes are attributed to an increase in the chymotryptic-like activity of trypsin, presumably as a result of dilution affecting the kinetics of the digestion reaction. We therefore investigated various types of digestions, including the use of an immobilized enzyme and on-column digestions. As a result of these studies, we developed conditions that produced a profile similar to typical microgram scale tryptic maps. These conditions involved the digestion of submicrogram amounts of rhGH in low microliter volumes with the addition of 10%trypsin. The small digest volume was achieved by lyophilization, in a siliconized Eppendorf tube, of the fraction collected from the secondary RP-HPLC step (typically 0.5-1 mL). At this point, most of the sample is irreversibly adsorbed onto the walls of the tube, but enzymatic (14)Riggin, A;Regnier, F. E. Anal. Chem. 1991,63, 468-474. (15) Cobb, K A; Novotny, M. V. Anal. Chem. 1992,64, 879-886.
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digestion can still be carried out to release the peptides in high yield for subsequent analysis. Even with careful optimization of these conditions, some loss of the more hydrophobic peptides can be expected, but this may be allowed for with suitably designed controls. Other digestion approaches did not give as good results. Metabolism Studies with Myeloleukemic Cells. Colosi et al. have developed a cell line in which the growth hormone receptor was expressed in a murine myeloleukemic cell line. In this system, small amounts of added growth hormones (low ng/ mL) result in cell proliferation as measured by the uptake of tritiated th~midine.~ We set up a study to isolate rhGH and its potential metabolites from such a cell-based system. rhGH was expected to bind to the cell receptors, to be internalized, and to be subsequently metabolized in the lysosomes. Ultimately the metabolites are secreted into the medium, from which we isolated any components that would bind to the affinity column. This system also contained fetal calf serum and other media components and, when coupled with the 24 h incubation time, produced a realistically demanding model on which to test the purification scheme. We found that rhGH was captured in high yields (approximately 90%)and with a high level of purity. The sample was characterized by tryptic mapping, and the results showed that small but significant amounts of deamidation (approximately 5%) had occurred in the sample that was incubated with cells for 24 h. The same reaction was observed in the control sample, and this is consistent with previous observationsthat growth hormone deamidates readily in neutral pH solutions.16 In contrast, a new peak at 57 min present in the sample incubated with cells was not present in the control nor in the tryptic map of native rhGH. The retention time of this peptide corresponds to that of tryptic fragment T1, which is missing the first two N-terminal amino acids (phenylalanine and proline). This peptide has been previously observed in the tryptic map of degraded rhGH and has been shown to be the result of a chemical process.17 This result will require further study but does demonstrate that this capture approach can be used to isolate rhGH and potential metabolites at low concentrations from a large volume of complex biological solution and then to identify a chemically modified form. The absence of extensive degradation of rhGH in this system is probably due to the smaller number of receptors (approximately 1000/cell) compared to the number in the liver (approximately 10 000/cell). Therefore, future studies have to be performed with cells that have a higher number of receptors or by using lower rhGH concentrations, e.g., 10 ng/mL. Growth Hormone Stability in Rat Serum. To prepare for in vivo experiments, we first isolated and purified rhGH added to rat serum. Additionally, this experiment provided information on the stability of growth hormone in serum in the absence of in vivo complications caused by cellular interactions and rapid clearance rates. Therefore, growth hormone (10 pg) was spiked into rat serum (1 mL) and isolated immediately (control) or incubated at 37 "C for up to 4 days. Alternatively, samples were spiked into PBS alone. These studies showed that growth hormone could be isolated in high yields (approximately70-80%) regardless of the incubation time or medium. No measurable (16) Johnson, B. A; Shivokawa, J. M.; Hancock, W. S.; Spellman, M. W.; Basa, L. J.; Aswad, D. M. J. Biol. Chem. 1989,264, 14262. (17) Battersby, J. E.; Hancock, W. S.; Canova-Davis, E.; Oeswein, J.; OConnor, B. Int. J Pept. Protein Res. 1994,44, 215-222.
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changes were observed in the tryptic maps of samples processed immediately. With increasing time of incubation, deamidation increased to 21% after 3 days of incubation in rat serum. This mod~cationis presumably chemical and not enzymatic, since a similar level was observed in the control sample (PBS alone). The longer incubation times were not particularly relevant to the planned in vivo study since the rapid clearance of growth hormone from the circulation dictated sampling to occur within 1h. This study did demonstrate that rhGH was relatively stable in this media, presumably due to the inactivation of proteolytic enzymes by inhibitors present in serum. In Vivo Oxidation. When we digested a small amount of rhGH and resolved the resulting fragments by capillary LC,we observed that the more hydrophobic peptides were recovered in poor yields. We therefore explored the addition of nonionic detergents (which do not significantly reduce the activity of trypsin) to the digestion mixture, in an effort to improve these recoveries. The addition of the detergent did improve recoveries signiiicantly, but it was found that even high-purity detergents, such as Tween 20, contained traces of peroxides, which at the very low sample amounts used in this study sometimes resulted in oxidation of methionine residues.18 Although the use of detergents can improve recoveries, their use is therefore not recommended when looking for evidence of oxidation. Additional studies also demonstrated that lesser amounts of oxidation could also occur as a result of sample handling, and the method described here has been designed to minimize sample degradation. While it is difticult to be certain of the exact extent of oxidation occurring in vivo compared to that occurring during sample preparation and the subsequent digest, validation of the purification scheme has shown that the affinity chromatography step combined with the secondary RPHPLC step did not induce oxidation. In addition, control digests in the absence of detergent indicated that artifactual oxidation only became significant at the submicrogram level. We therefore believe that the oxidation observed in a 18pg sample of rhGH, recovered at the 5 min time point, did occur in vivo and was not artifactual. The extent of in vivo oxidation occurring in the later time point samples is unclear, but a comparison of the tryptic maps clearly shows the trend of increasing amounts of oxidation with time. CONCLUSIONS
In this study we have demonstrated that a combination of affinity chromatography, W-HPLC,and micro-LC peptide mapping can allow the isolation in high yields and characterizationof small amounts of unlabeled proteins from biological systems. In addition, we show the advantage of using a receptor protein to capture bioactive protein rather than the traditional approach of capturing immunoreactive protein. Such studies should be of particular value to the elucidation of subtle metabolic events that may occur in vivo after administration of a protein pharmaceutical. Using this system, we observed in vivo oxidation of rhGH, which may be of consequence in the determination of product specifications of innocuous versus deleterious product variants. Such an approach has clear advantages over conventional studies using radioactive labeling but does place severe demands on analytical systems, particularly in the area of sample handling and high(18)Riggin, R M.; Dorulla, G. K; Miner, D. J. Anal. Biochem. 1987,167, 199209.
sensitivity detection. Furthermore, this study has demonstrated the general principles of an analytical approach for the study of protein metabolism, in vivo, without radioactive labeling. ACKNOWLEDGMENT The authors thank Dr. Abbie Celniker and Mr. Brian Fletcher for valuable discussions on antigen-antibody interactions and advice on immunoafkity purification. We thank Dr. Brian Fendly
for supplying mono- and polyclonal antibodies to rhGH and Ms. Deborah Mortensen for preparing in vivo samples. Received for review July 25, 1994. Accepted October 13, 1994.e AC940739P Abstract published in Advance ACS Abstracts, November 15, 1994.
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