Bmlogwals (1991) 19, 311-316

A Simple Method to Detect Contaminating Peptides and Amino Acids in Large-scale Ganglioside Preparations Giuseppe Corona, Sonia Mattioli, Alessandro Di Martino* and Lanfranco Callegaro Advanced Technology Division. Fidla S p A. Via Ponte della Fabbnca 3/A. 35031 Abano Terme. Italy

Abstract. A reverse-phase hqu,d chromatography method was developed to analyse the presence of contam~nahng peptldes and amino acids in large-scale monoslaloganglioslde preparabons Samples were hydrolysed under controlled conditions and denvatized with phenylisothiocyanate. PTC-amino acids were then separated and ,dentlfied by HPLC. The sensitwlty of the method allowed detechon of a least 50 pmoles of amino ac,d per mg of ganghoside with excellent reproduc,blhty and httle or no interference of by-products derwed from hydrolysis of the glycosphingohpld The response was consistently hnear for each amMno acid w,thln the examMned analyt,cal range The ease and speed of performance make th~s method a useful tool for rapidly mon,tonng large-scale extraction and pur#lcat,on processes of bJologlcals obtained from natural sources Introduction Ganghosldes are sialic acid-containing glycosphingolipids virtually ubiquitous in vertebrate cell membranes. 1.2They are pharmacologically active and have been used to treat peripheral and central nervous systems neuropathies for several years2 -5 Because the primary source of gangliosides for experimental and clinical use is brain tissue, we have developed a new application of the phenylisiothmcyanate (PITC) precolumn amino acid derwatization of Bidlingmeyer et al. t h a t enables determination of amino acids or small peptides t h a t are derived from protein hydrolysis, and are ultimately present in large-scale preparations. 6 We evaluated the sensitivity, reproducibility and lipid interference of this method. Adding different known amounts of amino acid standard solution and two different synthetic peptides to a crystallized ganglioside solution allowed quantitative recovery. The samples containing the peptides and the ganglioside were also analysed colorimetrically for protein determination; the results were compared to those obtained by our analytical system. Methods Crystalline monomaloganglioside (GM1) was supplied by Fidla (Abano Terme, Italy). Triethylamine To whom correspondence should be addressed A.D.M. is currently a Guest Research Scmnhst of Laboratory of Central Nervous System Studms, NINDS, Nahonal Institutes of Health, 9000 Rockvllle Pike, Bldg 36/4A15, Bethesda, Maryland 20892, U S.A 1045-1056/91/040311+06 $03 00/0

(TEA), phenylisothiocyanate, constant boiling-point hydrochloric acid, amino acids-H standards and bovine serum albumin were obtained from Pierce (Rockford, Illinois). The sequences of the synthetic peptides were Leu-Arg-Ala-His-Ala-Val-Asp-Ile-AsnGly (peptide R) and Ile-Lys-Val-Ala-Val (peptide L), respectively. They were synthemzed using a Milligen 9050 peptide s y n t h e s i z e r (Milhpore, Bedford, Massachusetts). HPLC grade acetonitrile and Fohn phenol r e a g e n t were p u r c h a s e d from Merck (Darmstadt, Germany). High-purity distilled water was supplied by a Mlllipore MflliQ purification system. All other solvents and chemicals were reagent grade, or the highest purity commercially available. The colonmetric protein determinations were made by a standard Lowry assay using bovine serum albumin as reference protein 7

Sample preparation GM1 was resuspended in distilled water and different amounts of standard amino acid solution and peptides were added. Samples were then transferred into glass vials and lyophilized using a SVH-100 Speed Vac concentrator (Savant, Farmmgdale, New York). Gas-phase hydrolysis was performed under vacuum for 1 h at 157°C in vials containing 0.6 ml of 6 N hydrochloric acid. s Each sample was then dissolved in 0.1 ml of 30% (v/v) acetonitrile/1 mM HC1, centrifuged in a model 5415 table-top centrifuge (Eppendorf, Hamburg, Germany) for 10 min at 14 000 rpm at room temperature, filtered on Millipore O 1991 The international Assoclatmnof BiologicalStandardlzahon

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Millex-HV 0.45 pm cartridges to eliminate particulates and vacuum-dried again. Amino acid der~vat~zat~on and analys~s S a m p l e s were solubilized in 0.05 ml of ethanol/water/TEA 2:2:1 (v/v). They were dried by rotary evaporation and again dissolved in 0.05 ml of coupling buffer (ethanol/water/TEA/PITC 7:1:1:1). Coupling ran for 20 min at room temperature. They were then finally evaporated to dryness under 20 mbar vacuum and dissolved in 0.2 ml of 10 mM sodium phosphate buffer, pH 7.0, containing 10% (v/v) acetonitrile. HPLC equipment consisted of two Waters 510 pumps, a WISP 250 autoin3ector and an automated gradient controller (Millipore, Bedford, Massachusetts). It was matched to a Perkm-Elmer LC 95 UV-Vis Detector (Norwalk, Connecticut). Twenty microlitre samples (10% of total volume were injected onto a C18 reverse-phase HPLC Beckman Ultrasphere ODS 250 × 4.6 mm (San Ramon, California) packed with 5 pm spherical particles and kept at a constant temperature of 40°C by a Pharmacm $5/20 heating block (Uppsala, Sweden) Eluents were' la) 0.14 M sodmm acetate containing 0-5c~, (v/v) of TEA, 6~ (v/v) acetonitrile and titrated to pH 6-35 with glacml acetic acid; and (b) 60% (v/v) acetonitrile. Eluents were constantly blanketed w~th helium by a Waters eluent stabilizatmn system. The separation gradient consisted of a 15 min run from 0c~ to 30ok of buffer (b), 15 min to 45% of(b) then 10 rain to 100% of(b). The column was washed for 5 min with 100% of(b) and re-equilibrated to 100% (a) in 15 mm. Column flow was 1.0 ml/min and runs were monitored at 269 nm. Chromatographic data were elaborated by a Waters 820 software data-processing system. The amino acid analyser was a model 3A29 matched to a 6 DC durum gel 250 × 4.6 mm column (Carlo Erba, Milano, Italy). Analyses were performed under standardized experimental conditions2 .1° Elution buffers were 0.15 M sodium citrate, pH 3-3, and 1 2 M sodium citrate, pH 6.8. Results

Figure 1 shows HPLC profiles obtained after acid hydrolysis and PITC derivatization Wig. 1(al-a3)]. A1 represents a chromatography run of a sample containing 1 mg of GM1. The two major peaks, design a t e d U1 a n d U2, respectively, r e p r e s e n t e d unidentified degradation products which resulted from lipid hydrolysis. The third peak marked PTU, was assigned to phenylthiourea. This is a slde-reac-

tlon product formed during derivatization, and easily identified by monitoring a blank run of the derivatization mixture in which neither ganglioside nor amino acids were added (data not shown). A single peak corresponding to the decomposition of the glycosphingohpid was observed in the same sample evaluated by the amino acid analyser [Fig. l(bl)]. One nanomole and 10 nanomoles of amino acid standard were then added to the same amount of GM1. The mixtures were derivatized, and the chromatographic elutions are shown, respectively, at [Fig. l(a2 and a3)]. All denvatized amino acid peaks were resolved and easily identified in both samples, and integrated values of PTC-amino acid peak areas mdmated t h a t the two samples were quantitatively correlated. Comparable results were also obtained when 0.5 nanomoles of amino acids were added to GM1. This was the lower level of sensitivity for our experimental conditions since reduced amounts of standard yielded mgnals indistinguishable from the background of the blank run. Figure l(b2 and b3) show the same samples as in Fig. 1 (a2 and a3) when evaluated by the amino acid analyser. No amino acid was detected in the sample containing 1 nanomole of standard; additionally, 10 nanomoles of standard were still below the limit of detection. Increasing amounts of GM1 and standard injected were still below the limit of detection. Increasing amounts of GM1 and standard injected into the analyser column yielded complicated elution profiles where interferences from lipid hydrolysis prevented quantitative determination (data not shown). The response factors and linearity for each amino acid were evaluated in the range of 0.6-10 nanomoles m the presence of 1 mg of GM1. These data are summarized in Table 1. All amino acids except cysteine had comparable response factors. Thin amino acid is m fact destroyed during the hydrolysis step prior to the derivatization. The reproducibility of quantitative data was calculated by adding 1 and 10 nanomoles of amino acids to 1 mg of GM1, and is illustrated in Fig. 2. Each plot represents the average recovery for each amino acid after seven independent determinations Disregarding values for cysteine, the only values showing a relatively greater standard deviation in the sample with the lower amino acid standard concentration were PTC-phenylalanine, proline and alanine. The average recovery for all amino acids was 0-713 + 0-030 nanomoles when samples containing 10 nanomoles of standard were injected in the column in the presence of GM1 concentrations varying between 0.1 and 5 mg This recovery was practically

Detecting contaminating pepUdes and amino acids

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F i g u r e 1. Elutlon profiles after PITC demvatization of 1 mg GM1 (al), 1 mg of GM1 spiked, respectively, with 1 nanomole (a2) and 10 nanomoles (a3) of amino acid standard. The minor unidentified peaks appearing in the background of the blank GM1 run (al) represent decompomtion products of the glycosphlngolipld matrix. The corresponding samples eluted from the column of the amino acid analyzer are shown m bl-b3. Ten percent of the sample volume was analysed in each determination. c o n s t a n t a n d not influenced by t h e p r e s e n c e of t h e lipid within this c o n c e n t r a t i o n r a n g e (Fig. 3). T h e exception h e r e w a s the r e c o v e r y of t y r o s i n e which w a s lower t h a n expected. T h e detailed e v a l u a t i o n of this p a r a m e t e r i n d i c a t e d its c o n s t a n t d e c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s of monosialoganglioside. B o t h curves s t e e p l y i n c r e a s e d w h e n the a m o u n t of GM1 w a s i n c r e a s e d to 10 a n d 20 mg.

Vamable k n o w n a m o u n t s of p e p t i d e s were finally i n t r o d u c e d into a s a m p l e c o n t a i n i n g 1 m g of GM1, a n d were s u b s e q u e n t l y d e t e r m i n e d e i t h e r by PTCa m i n o acid d e r i v a t i z a t i o n or L o w r y assay. D a t a o b t a i n e d by P T C - a n a l y s e s w e r e corrected according to the r e s p o n s e factors which were described above (see Table 1), a n d are s h o w n in Table 2 t o g e t h e r with those calculated colorimetmcally. Both d e t e r m i n a -

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F i g u r e 2. Values of data reproducibility for each amino acid as evaluated by adding to 1 mg of GM1 1 nanomole (a) and 10 nanomoles (b) of amino acld standard solution. T a b l e 1. R e s p o n s e f a c t o r a n d h n e a r c o r r e l a t i o n c o e f f i c i e n t f o r e a c h a m i n o a c i d in q u a n t i t a t i v e d e t e r m i n a t i o n b e t w e e n 0.6 a n d 10 n a n o m o l e s A m i n o acid

R e s p o n s e factor":

Ala Arg Asp Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val

3.11 3.84 3.78 3.49 3-07 3.96 2.86 3.17 2-01 1.03 3.39 4-10 4.10 4.91 4.67 3.00

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t]ons of p e p t i d e s L a n d R w e r e c a l c u l a t e d w i t h a n a v e r a g e e r r o r of +4.0% b y t h e H P L C m e t h o d . However, different v a l u e s w e r e o b t a i n e d w h e n determ i n a t i o n s w e r e m a d e w i t h the L o w r y assay. S a m p l e s c o n t a i n i n g t h e ganglioside a n d d e c a p e p t i d e R indicated a n i n a c c u r a t e d e t e r m i n a t i o n w h e n less t h a n 5.0 p g w e r e p r e s e n t in the s a m p l e a n d o v e r e s t i m a t e d its p r e s e n c e at h i g h e r c o n c e n t r a t i o n s . Conversely, valu e s for p e n t a p e p t i d e L w e r e below a n y level of significance.

r2

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0.998 0.999 0.992 0-996 0.997 0.998 0.999 0.959 0.987 0.996 0.923 0.999 0.996 0 994 0.999 0.997

Discussion

' Response factors are calculated m plcomoles x pV ~3 s 1.

T h e c u r r e n t p h a r m a c e u t i c a l use ofbiologicals derived f r o m n a t u r a l sources ~mplies t h a t t h e r a p e u t i c s for h u m a n u s e m u s t be purified first in c o m p l i a n c e w i t h guidelines issued by r e g u l a t o r y agencies. In p a r t i c u lar, t h e y m u s t be devoid of a n y foreign c o n t a m i n a n t s , such as p r o t e i n s of p e p t i d e s f r o m d i s r u p t i o n of tiss u e s occurring d u r i n g t h e p r e p a r a t i o n p r o c e d u r e s a n d t h a t m i g h t c o n s t i t u t e a h a z a r d to h u m a n s . 1]

T a b l e 2. P e p t i d e d e t e r m i n a t i o n by L o w r y a s s a y a n d P I T C d e r i v a t i z a t i o n in the p r e s e n c e of GM1 Peptide R Amount spiked (pg)

Peptide L

PTC amino acid (pg)

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__ 0-8 2.3 4.0 6.9 15.2

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0.5 1.0 2.0 4-0 8.0 16-0 NS, not significant.

Detecting contaminating peptides and amino acids

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Figure 3. Total average recovery of amino acids (--O--) and recovery of tyrosine (--A--) at varying amounts of GM1. Detecting proteins and peptides as contaminants in large-scale preparations of products extracted from natural sources relies on the widespread use of colorimetric assays. They are widely accepted because they are economical and easy to perform. 7'12However, these methods all lack specificity, stability of some reagents, have low sensitivity in the determination of short amino acid sequences and a poor tolerance towards interference caused by the presence oflipids or reducing agents. I2-14 There are a few methods to determine low amounts ofpeptides and proteins, but they rely on expensive chemicals and radioactive compounds. 15 PITC derivatization is one of the most commonly used pre-column techniques for amino acid determination, s The coupling reaction does not yield multiple side-derivatives, labelled amino acids derivatives are sufficiently stable at room temperature and can be easily analysed by HPLC monitoring at the appropriate wavelength. The system we have adapted has a sensitw~ty t h a t can detect as little as 50 nanomoles of amino acid with excellent resolution and can also yield quantitative data within a wide analytical range. Close evaluation and comparison of the chromatography profiles indicated t h a t the retention time of peak U1 was different from t h a t of any PTCamino acid, and did not interfere with the determinations. On the other side, peak U2 was relatively close to t h a t of PTC-phenylalanine, and the fact t h a t this peak was not completely resolved at low standard concentration accounted for its low linear correlation coefficient. The higher standard dewation values observed particularly for alanine and proline

315

at low concentration of standard, can also be explained by their limited resolution from some of the other minor peaks constituting the background of the black run. The entire system was not significantly affected by concentrated lipids in the sample. Up to 5 mg of GM1 in the sample did not affect the quantitative recovery of the amino acids. When the amount of ganglioside was i n c r e a s e d , t h e b a c k g r o u n d p e a k s predommated and the integrator could no longer resolve them from those of the real PTC-amino acids. An interesting observation was made for tyrosine, whose PTC-amino acid peaks did not fall near any of the background peaks, but whose recovery was consistently dependent on ganglioside concentration. This is probably due to non-specific interaction with degradation products from the hydrolysed matrix, and remains unexplained. The comparison of our methodology with the Lowry assay also deserves consideration. Our method ~s based on the absolute determination of the amino acids and gives reproducible results in detecting both peptides. Conversely, it has been shown that the Lowry method may yield unreliable data below 5 pg of protein.12 This was confirmed when samples containing the decapeptide were analysed. The presence of the amino acid sequence was overestimated at a higher concentration; this phenomenon has also been observed when different peptides or even proteins were analysed in the presence of GM1 (unpublished observation). The smaller peptide was totally undetectable. The main problem of detecting residual contamination by peptides or amino acids as contaminants derived from protein disruption is related to the presence of small fragments formed by shearing of the larger proteins. We think t h a t th~s methodology could be integrated with other techniques currently used for the analyses of purified products in extraction processes ofbiologicals from natural sources to estabhsh to the highest degree t h a t these products consistently meet the required integrity, identity and purity.

Acknowledgments The authors t h a n k Devera G. Schoenberg for editing the manuscript and M. Cristina Bogoni for document production assistance. References

1. Wlegandt H Gangliosldes In: Wiegandt H, ed. Glycolipids New Comprehenswe Bmchemlstry, Vol. 10. Amsterdam, The Netherlands" Elsevier 1985, 199-260.

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2 Yu RK, Salto M. Structure and localization of gangliosides. In: Margohs RU, Margolis RK, eds. Neuroblology of Glycoconjugates. New York. Plenum Press, 1989; 1-42. 3. Sabel BA, Stem DG. Pharmacological treatment of central nervous system injury Nature 1986; 323. 493. 4. Trlban C, Guidohn D, Fabris M, Marlin P, Schiawnato A, Don~ M, Bortolami MC, Di Glamberardmo L, Flori MG. Ganglioslde treatment and improved axonal regeneration capacity m experimental diabetic neuropathy. Diabetes 1989; 38: 1012-1022. 5. Ge~sler FH, Dorsey FC, Coleman WP. Motor recovery following human spinal cord injury. A randomized placebo controlled tmal with GM1 ganglioside. N Engl J Med 1991; 324: 1829-1838. 6. Bidhngmeyer BA, Cohen SA, Tarvm TL. Rapid analysis of amino acids using pre-column derlvatlzatlon. J Chromatogr 1984; 336 93-104. 7. Lowry OH, Rosebrough NJ, Farr AL, Randall KJ. Protein measurement with the Fohn phenol reagent. J Bml Chem 1951; 193: 265-271. 8 Meltzer NM, Tous GI, Gruber S, Stein S. Gas-phase hydrolysis of proteins and peptldes. Anal Biochem 1987; 160" 356-361. 9. Moore S, Spackman DH, Stem WH. Chromatography of amino acids on sulfonated polystyrene resins An improved system. Anal Chem 1958; 30' 1185-1190.

10. Hamilton PB. Ion exchange chromatography of amino acids. A single column, high resolving, fully automatm procedure. Anal Chem 1963; 35. 2055-2061. 11 Committee for Proprmtary Medicinal Products: ad hoc working party on blotechnology/pharmacy and working party on safety of medmmes. Notes to applicants for marketing authorizations on the pre-clinical biological safety testing of medicinal products derived from blotechnology (and comparable products derived from chemical synthesis). J Biol Stand 1989; 17. 203-212. 12. Peterson GL. Review of Fohn phenol protein quantitation of Lowry, Rosebrough, Farr and Randall. Anal Blochem 1979; 100: 201-212. 13. Chou SC, Goldstein A. Chromogenic groupings in the Lowry protein determinations Blochem J 1960, 75. 109-115. 14. Emhberg J, Mokrasch LC. Interference by oxidized liplds in the determination of protein by the Lowry procedure. Anal Biochem 1969.30.386-390. 15 Nishio K, Kawakaml M. Protein assays in very diluted solutions. Anal Biochem 1982; 126: 239-341.

Received for publication 24 May 1991; accepted 27 August 1991.