RESUMO
Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For example, high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.
Assuntos
Imunoglobulina G/química , Oligossacarídeos/isolamento & purificação , alfa-L-Fucosidase/química , beta-Galactosidase/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicosilação , Humanos , Hidrólise , Imunoglobulina G/sangue , Oligossacarídeos/química , Reprodutibilidade dos TestesRESUMO
One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.
Assuntos
Glicoproteínas/metabolismo , Calicreínas/metabolismo , Polissacarídeos/metabolismo , Antígeno Prostático Específico/metabolismo , Cromatografia Líquida , Glicosilação , Humanos , Laboratórios , Espectrometria de Massas/métodos , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.
Assuntos
Asparagina/química , Cromatografia Líquida de Alta Pressão , Ácido N-Acetilneuramínico/análise , Oligossacarídeos/análise , ortoaminobenzoatos/química , Animais , Sequência de Carboidratos , Bovinos , Cromatografia Líquida de Alta Pressão/normas , Fetuínas/química , Fetuínas/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/normas , Oligossacarídeos/normas , Orosomucoide/química , Orosomucoide/metabolismo , Padrões de ReferênciaRESUMO
Glycoprotein sialylation analysis is a common analytical step in characterizing biotherapeutic products and expression experiments to optimize production. In this article, a high-throughput (5-min) high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD)-based analytical method for glycoprotein sialic acid determination is described. Results from this method are compared with both published HPAE-PAD and 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by ultra high-performance liquid chromatography fluorescence detection (UHPLC-FLD) assays. The quantified sialic acid amounts agree with prior HPAE-PAD analyses within replicate error and with UHPLC-FLD within an average of 24%, which are equivalent results based on assay reproducibility.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Glicoproteínas/análise , Ácidos Siálicos/análise , Animais , Calibragem/normas , Glicoproteínas/química , Ensaios de Triagem em Larga Escala , Humanos , Mamíferos , Fenilenodiaminas/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ácidos Siálicos/química , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Many important vaccines use bacterial capsular polysaccharides, or shorter polysaccharides or oligosaccharides, derived from the capsular polysaccharides, conjugated to protein. It is imperative that manufacturers understand the carbohydrate composition of these vaccines and deliver a product with a consistent polysaccharide or polysaccharide conjugate composition and content. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a major technique used to understand the carbohydrate composition of these vaccines and ensure product quality. HPAE-PAD separates and detects carbohydrates without analyte derivatization. This paper describes the basics of the HPAE-PAD technique and then reviews how it has been applied to Haemophilus influenzae type b, pneumococcal, meningococcal, group B streptococcal, and Salmonella polysaccharide and corresponding conjugate vaccines.
Assuntos
Cromatografia por Troca Iônica/métodos , Eletroquímica/métodos , Vacinas/normas , Ânions , Monossacarídeos/isolamento & purificação , Polissacarídeos/análiseRESUMO
We developed and validated an ion chromatography method to assay iodide in serum sampled from rats and rabbits that had been exposed to iodomethane. Iodomethane is of interest because it is a volatile liquid pre-plant soil crop protection fumigant that has been proposed as a non-ozone-depleting alternative to methyl bromide. Serum was prepared from whole blood collected on wet ice at the time of sacrifice and kept frozen at less than -65 degrees C. For analysis, serum samples were thawed unassisted at ambient temperature. Proteins were separated from the serum samples by ultrafiltration. A 100-microl filtered serum sample was then injected into the ion chromatograph without additional sample preparation. Iodide was separated in <20 min by anion-exchange chromatography using a 25-mM nitric acid eluent. The analyte of interest was detected by pulsed amperometry using a silver working electrode. The method showed linear response over the concentration range of 100 to 5000 ng/ml iodide (r2>.998) with a lower limit of quantitation of 100 ng/ml iodide. The accuracy of the procedure, determined by spiked recovery measurements at 100 ng/ml iodide, was between 90 and 110%. A method detection limit of 20 ng/ml for iodide in serum samples was demonstrated using the method of standard additions.
Assuntos
Iodetos/sangue , Animais , Cromatografia por Troca Iônica/instrumentação , Cromatografia por Troca Iônica/métodos , Cromatografia por Troca Iônica/normas , Eletroquímica/instrumentação , Eletroquímica/métodos , Eletroquímica/normas , Hidrocarbonetos Iodados/análise , Hidrocarbonetos Iodados/sangue , Iodetos/análise , Masculino , Coelhos , Ratos , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização por Electrospray/normasRESUMO
Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.
Assuntos
Antibacterianos/análise , Cromatografia por Troca Iônica/métodos , Eletroquímica/métodos , Canamicina/análise , Nebramicina/análise , Neomicina/análise , Tobramicina/análise , Antibacterianos/química , Contaminação de Medicamentos/prevenção & controle , Fermentação , Concentração de Íons de Hidrogênio , Canamicina/isolamento & purificação , Nebramicina/isolamento & purificação , Neomicina/isolamento & purificação , Controle de Qualidade , Padrões de Referência , Streptomyces/metabolismo , Tecnologia Farmacêutica , Tobramicina/químicaRESUMO
Cyanide is a regulated contaminant in drinking water in the United States. This paper describes an ion chromatography method with pulsed amperometric detection (PAD) that directly determines free cyanide in drinking water. Samples are treated with sodium hydroxide to stabilize cyanide and with a cation-exchange cartridge to remove transition metals. Cyanide is separated by anion-exchange chromatography and detected by PAD with a waveform optimized for cyanide and used with a disposable silver working electrode. The recovery of cyanide spiked into five water samples was >80%. With an MDL of 1.0 microg/L, this method determines cyanide concentrations well below the reporting limits for free cyanide in drinking water.
Assuntos
Cromatografia por Troca Iônica/métodos , Cianetos/análise , Abastecimento de Água/análise , Cromatografia por Troca Iônica/instrumentação , Eletrodos , Reprodutibilidade dos Testes , Estados UnidosRESUMO
Neomycin B is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that neomycin sulfate and its major impurities, including neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.40 mM) at a column temperature of 30 degrees C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between neomycin B and the closest major impurity ranged from 6.56 and 7.45 over 10 days of consecutive analysis (7.24+/-0.10, n=836 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (10 days, 822 injections) retention time stability with a R.S.D. of 0.6%. Peak area R.S.D. (10 microM) was 1.3%. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. The spike recoveries of neomycin B from extractions of three different topical ointments and cream formulations ranged from 95 to 100%. The measured concentration of neomycin B in these formulations ranged from 119 to 154% of the label concentration. The R.S.D. for the measured concentration of one of the formulations tested over three separate days, n=11 extracts, was 3.2%. Based on the results of these evaluations, we believe this method can be used for neomycin sulfate identity, assay, and purity.
Assuntos
Antibacterianos/análise , Neomicina/análise , Aminoglicosídeos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Contaminação de Medicamentos , Eletroquímica , Framicetina/análise , Pomadas , Padrões de Referência , Reprodutibilidade dos Testes , TemperaturaRESUMO
Tobramycin is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that tobramycin and its major impurities, including kanamycin B and neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.00 mM) at a column temperature of 30 degrees C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between tobramycin and kanamycin B ranged from 5.71 and 6.06 over 7 days of consecutive analysis (5.92+/-0.07, n = 590 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (50 days, 2368 injections) retention time stability with R.S.D.s of 0.4% and 0.3% for tobramycin and kanamycin B, respectively. Peak area R.S.D.s for tobramycin and kanamycin B (10 microM each, 20 microL injection) over 7 days (572 injections) were 2.3% and 1.9%, respectively. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. Based on the results of these evaluations, this method can be used for tobramycin identity, assay, and purity.
Assuntos
Antibacterianos/análise , Tobramicina/análise , Resinas de Troca Aniônica , Cromatografia por Troca Iônica , Eletroquímica , Eletrodos , Ouro , Canamicina/análogos & derivados , Canamicina/análise , Reprodutibilidade dos Testes , Tecnologia Farmacêutica , TemperaturaRESUMO
Anion-exchange chromatography with integrated pulsed amperometric detection (AE-IPAD) separates and directly detects amino acids, carbohydrates, alditols, and glycols in the same injection without pre- or post-column derivatization. These separations use a combination of NaOH and NaOH/sodium acetate eluents. We previously published the successful use of this technique, also known as AAA-Direct, to determine free amino acids in cell culture and fermentation broth media. We showed that retention of carbohydrates varies with eluent NaOH concentration differently than amino acids, and thus separations can be optimized by varying the initial NaOH concentration and its duration. Unfortunately, some amino acids eluting in the acetate gradient portion of the method were not completely resolved from system-related peaks and from unknown peaks in complex cell culture and fermentation media. In this article, we present changes in method that improve amino acid resolution and system ruggedness. The success of these changes and their compatibility with the separations previously designed for fermentation and cell culture are demonstrated with yeast extract-peptone-dextrose broth, M199, Dulbecco's modified Eagle's (with F-12), L-15 (Leibovitz), and McCoy's 5A cell culture media.
Assuntos
Aminoácidos/análise , Meios de Cultura/metabolismo , Acetatos , Aminoácidos/isolamento & purificação , Carboidratos/isolamento & purificação , Cromatografia por Troca Iônica , Fermentação , Lisina/isolamento & purificação , Álcoois Açúcares/isolamento & purificação , Fatores de TempoRESUMO
Sucralose is a chlorinated carbohydrate nonnutritive sweetener of food and beverage products. The determination of sucralose in food and beverages is important to ensure consistency in product quality. Sucralose was determined in two commercial products without sample preparation using high-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD). Sucralose was determined with a 10 min isocratic separation. To determine sucralose and other carbohydrates (e.g., dextrose) simultaneously, a gradient separation was developed. The linear range of electrochemical response extended over 3 orders of magnitude, from 0.01 (LOD) to 40 microM (16 microg/mL; 25 microL injection). High precision, high spike recovery, and method ruggedness were observed for both samples.
Assuntos
Bebidas/análise , Cromatografia Líquida de Alta Pressão/métodos , Sacarose/análogos & derivados , Sacarose/análise , Edulcorantes/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
An ion chromatography (IC) method was developed for the simultaneous determination of total nitrogen and total phosphorus after alkaline persulfate digestion. This study takes advantage of advances in construction of high-resolution, high-capacity anion-exchange columns that can better tolerate the matrices typically encountered when a determination of total nitrogen and total phosphorous is required. Here, we used an electrolytically generated hydroxide eluent combined with a high-capacity, hydroxide-selective, anion-exchange column for the determination of total nitrogen (as nitrate-N) and total phosphorus (as phosphate-P) in environmental samples by IC. This method yielded LODs for nitrate-N and phosphate-P of 1.0 and 1.3 µg/L, respectively. The LOQs determined for these analytes were 3.4 and 4.2 µg/L, respectively. Due to the dilution factor required and the blank nitrate-N concentration after the persulfate digestion, the quantification limits increased for nitrate-N and phosphate-P to 171 and 63 µg/L, respectively. The suitability of the method was evaluated by determining the nitrogen and phosphorus concentrations from known concentrations of organic-containing nitrogen and phosphorus compounds. In addition, environmental samples consisting of six different wastewaters and 48 reservoir samples were evaluated for total nitrogen and phosphorus. The recoveries of nitrogen and phosphorus from the organic-containing compounds ranged from 93.1 to 100.1% and 85.2 to 97.1%, respectively. In addition, good correlation between results obtained by the colorimetric method and IC was also observed. The linearity, accuracy, and evaluation of potential interferences for determining TN and TP will be discussed.
Assuntos
Cromatografia por Troca Iônica/métodos , Nitratos/análise , Nitrogênio/análise , Fósforo/análise , Sulfetos/química , Água/química , Álcalis/química , Cromatografia por Troca Iônica/instrumentação , Limite de DetecçãoRESUMO
The United States Pharmacopeia USP35-NF30 contains monographs for adenosine and sodium bicarbonate with imprecise and outdated color comparison assays for ammonia. In the spirit of monograph modernization we developed ion chromatography (IC) assays for ammonia that meet the requirements of the adenosine and sodium bicarbonate monographs. Ammonia, as ammonium, in a solution of adenosine is separated on a high-performance cation-exchange column and detected by suppressed conductivity. This assay requires <5min per sample and accurately determines the ammonia content of adenosine. For sodium bicarbonate a different IC method was required to determine ammonia due to the high concentration of sodium relative to ammonia in a sodium bicarbonate solution. This assay uses a high-capacity cation-exchange column that has the appropriate selectivity for determining low concentrations of ammonia in the presence of high concentrations of sodium. For both the methods, the precisions (retention time RSD<0.1%, peak area RSD<2.3%), accuracy (average recovery 70-117), limits of detection (LOD) and quantitation (LOQ), and robustness were measured according to the analytical performance requirements described in USP General Chapter <1225>.
Assuntos
Amônia/análise , Química Farmacêutica/tendências , Limite de Detecção , Preparações Farmacêuticas/análise , Química Farmacêutica/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia por Troca Iônica/tendênciasRESUMO
The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Neomicina/análise , Antibacterianos/análise , Antibacterianos/química , Formas de Dosagem , Neomicina/química , Farmacopeias como Assunto , Reprodutibilidade dos Testes , Fatores de Tempo , Estados UnidosRESUMO
Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.
Assuntos
Cromatografia por Troca Iônica/métodos , Eletroquímica/métodos , Triptofano/análise , Ração Animal , Animais , Bovinos , Meios de Cultura/química , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Saccharomyces cerevisiae/química , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Hidróxido de Sódio/farmacologiaRESUMO
Cell culture and fermentation broth media are used in the manufacture of biotherapeutics and many other biological materials. Characterizing the amino acid composition in cell culture and fermentation broth media is important because deficiencies in these nutrients can reduce desired yields or alter final product quality. Anion-exchange (AE) chromatography using sodium hydroxide (NaOH) and sodium acetate gradients, coupled with integrated pulsed amperometric detection (IPAD), determines amino acids without sample derivatization. AE-IPAD also detects carbohydrates, glycols, and sugar alcohols. The presence of these compounds, often at high concentrations in cell culture and fermentation broth media, can complicate amino acid determinations. To determine whether these samples can be analyzed without sample preparation, we studied the effects of altering and extending the initial NaOH eluent concentration on the retention of 42 different carbohydrates and related compounds, 30 amino acids and related compounds, and 3 additional compounds. We found that carbohydrate retention is impacted in a manner different from that of amino acid retention by a change in [NaOH]. We used this selectivity difference to design amino acid determinations of diluted cell culture and fermentation broth media, including Bacto yeast extract-peptone-dextrose (yeast culture medium) broth, Luria-Bertani (bacterial culture medium) broth, and minimal essential medium and serum-free protein-free hybridoma medium (mammalian cell culture media). These media were selected as representatives for both prokaryotic and eukaryotic culture systems capable of challenging the analytical technique presented in this paper. Glucose up to 10mM (0.2%, w/w) did not interfere with the chromatography, or decrease recovery greater than 20%, for the common amino acids arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamate, aspartate, cystine, and tyrosine.