Reduction of Adsorption
20230041240 · 2023-02-09
Assignee
Inventors
- Andreas Geissler (München, DE)
- Kristina Kemter (Garching, DE)
- Sabine Hauck (München, DE)
- Maximilian Maarten Böckel (Martinsried, DE)
Cpc classification
C07K17/02
CHEMISTRY; METALLURGY
A61J1/1468
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a method of reducing or preventing the adsorption of a polypeptide on a surface, or the aggregation of a polypeptide in a liquid composition in contact with a surface comprising the steps of a) providing a composition comprising the polypeptide and at least two different amino acids; b) contacting the composition with the surface. Furthermore, the invention relates to medical devices comprising a composition comprising the polypeptide and at least two different amino acids.
Claims
1. A method of reducing or preventing the adsorption of a polypeptide on a surface comprising the steps of: a) providing a composition comprising the polypeptide and at least two different amino acids; b) contacting the composition with the surface.
2. A method of reducing or preventing the aggregation of a polypeptide in a liquid composition in contact with a surface, wherein the method comprises the steps of: a) providing a composition comprising the polypeptide and at least two different amino acids; b) contacting the composition with a surface.
3. The method of any of the preceding claims, wherein the composition comprises at least three different amino acids selected from three different groups of the groups consisting of: (a) amino acids with non-polar, aliphatic R groups; (b) amino acids with polar, uncharged R groups; (c) amino acids with positively charged R groups; (d) amino acids with negatively charged R groups; and (e) amino acids with aromatic R groups.
4. The method of any of the preceding claims, wherein the composition comprises at least five amino acids from five different groups.
5. The method of any of the preceding claims, wherein the composition is an aqueous solution.
6. The method of any of the preceding claims, wherein the composition is free or substantially free of at least one stabilising protein and/or wherein the composition is free or substantially free of at least one surfactant.
7. The method of any of the preceding claims, wherein the polypeptide concentration in the composition is at least 100 mg/ml, preferably at least 150 mg/ml.
8. The method of any of claims 1 to 8, wherein the polypeptide concentration in the composition is not higher than 20 mg/ml, preferably not higher than 5 mg/ml.
9. The method of any of the preceding claims, wherein the polypeptide is comprised or attached to the capsid or envelope of a virus or a viral vector.
10. The method of any of the preceding claims, wherein the surface comprises or consists of a metal, a glass or a polymer material, preferably selected from polyethylene (PE), for example selected from polyethylene terephthalate (PET), high density polyethylene (HDPE), low-density polyethylene (LDPE), linear low density polyethylene (LLDPE), polypropylene (PP), polystyrene (PS), polyvinylchloride (PVC), polyvinylidene chloride (PVDC), polytetrafluorethylene (PTFE), polyethersulphone (PES), polymethylmethacrylate, polycarbonate (PC, bisphenol A), nylon, polyether urethane, polysiloxane (silicone), polychlorotrifluoroethylene (PCTFE)/PVC laminates, polyamide (PA), e.g. orientated polyamide (OPA), cyclic olefin copolymer, or mixtures and copolymers thereof. The polymer may further be natural or synthetic elastomer such as for example latex, polyisoprene rubber, chloroprene (2-chloro-1,3-butadiene), styrene-butadiene rubbers (SBR) silicone rubber, and butyl rubber.
11. The method of any of the preceding claims, wherein the surface is a hydrophobic surface.
12. The method of any of the preceding claims, wherein the surface is part of a medical device.
13. A method of administering a polypeptide to a subject using a medical device, wherein a composition comprising a polypeptide is exposed to a surface of the medical device wherein the polypeptide is administered in a composition according to the preceding claims.
14. A composition comprising a polypeptide and at least at least two different amino acids according to the preceding claims for use in a method of treating a patient wherein said composition is exposed to a surface of a medical device during administration of the composition to the patient.
15. A medical device comprising a composition comprising a polypeptide and at least at least two different amino acids according to the preceding claims.
16. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from group (a) of amino acids with an unpolar aliphatic R group; and at least one amino acid is selected from group (c) amino acid with a positively charged R group.
17. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from group (c) of amino acids with positively charged R groups; and at least one amino acid is selected from group (e) of amino acids with aromatic R groups.
18. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from group (a) of amino acids with an unpolar aliphatic R group; and at least one amino acid is selected from group (e) of amino acids with aromatic R groups.
19. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from group (b) of amino acids with a polar uncharged R group; and at least one amino acid selected is from group (e) of amino acids with aromatic R groups.
20. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from group (d) of amino acids with negatively charged R groups; and at least one amino acid is selected from group (e) of amino acids acid with aromatic.
21. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least three different amino acids wherein the at least three amino acids are at least one amino acid selected from group (b) of amino acids a polar uncharged R, or at least one amino acid from group (a) of amino acids with an unpolar aliphatic R group, and further at least one amino acid is selected from group (d) of amino acids with negatively charged R groups; and at least one amino acid is selected from group (e) of amino acids with aromatic R groups.
22. The method, composition or device according to claims 1, 2, 5 to 15, wherein the composition comprises at least two different amino acids wherein at least one amino acid is selected from any of the groups of amino acids with (a) of amino acids with an unpolar aliphatic R group (b) of amino acids with a polar uncharged R group;(c) of amino acids with positively charged R groups; and/or (d) of amino acids with negatively charged R groups; and further comprises at least one amino acid selected is from group (e) of amino acids with aromatic R groups.
23. The method, composition or device according to any of the preceding claims, wherein the composition does not comprise arginine and/or lysine other than in the polypeptide.
Description
FIGURE LEGENDS
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[0148] The following examples exemplify the invention described afore.
Example 1: Surface Absorption of Anti-IL-6 Antibody
1.1. Materials and Methods
[0149] In order to directly analyze the reduction of surface adsorption of biomolecules particularly of proteins on solid surfaces by different amino acid mixtures an anti-IL6-ELISA was applied as a model for characterization and quantification of surface adsorption of an anti-IL6 coating antibody (lgG1) as modell protein. ELISA technology is a well known surface-sensitive technique to characterize the adsorption of biomolecules on surfaces with a high specificity and sensitivity and the possibility for automatization. ELISA is a technique that uses solid supports surfaces (multi-well plates or polystyrene beads) for the molecular quantification of immune-complexes via signal amplification through an enzymatic reaction readout. The reaction rates in all steps are limited by diffusion of the biomolecules generating therefore relatively long incubation times (h). In the ELISA assay used to analyse the reduction of surface adsorption according to the invention, adsorption of an anti-IL6 coating antibody on a hydrophobic polystyrene (PS) or polypropylene (PP) medium binding 96 well plate was directly quantified in the presence and absence of solutions in accordance with the liquid compositions of invention comprising and at least two different amino acids during coating of the wells with anti-IL6. The anti-IL6 antibody was diluted in different liquid composition prior the coating step. As comparison, the surface adsorption in the solutions according to the invention was compared to adsorption in 100 mM and 500 mM arginine*HCl as disclosed by Shikiya et al. (Shikiya Y,Tomita S, Arakawa T, Shiraki K. Arginine inhibits adsorption of proteins on polystyrenesurface (2013). PLoS One, 8 (8), e70762).
[0150] For quantification of the relative reduction of surface adsorption of the anti-IL6 coating antibody, the photometric absorption of positive reference samples using Blocking Buffer for dilution and incubation was defined as 100% reduction of adsorption and the photometric absorption of negative reference samples using PBS buffer or a low ionic strength 10 mM sodium phosphate buffer at pH 7.4 without any other additives for dilution and incubation was defined as 0% reduction of adsorption.
[0151] In detail, after dilution of the anti-IL6-antibody (monoclonal mouse IgG1; R&D systems Inc., USA) with PBS, Blocking Buffer (0.05 % Polysorbat 20, 0.01 % skim milk powder in PBS pH 7.4), solutions comprising arginine*HCl, or solutions comprising at least two amino acids according to the invention, coating of the antibody in the respective solutions was perfomred at 2 - 8° C. for 17 hours over night on polystyrene midium binding plates (Greiner Bio One™ 96-well Microtiter plates, Germany) or polypropylene plates (Eppendorf, Germany) at a anti-IL6 concentration of 1.5 \..Math.g/ml. After removal anti-IL6-antibody of solutions, the wells were incubated for one hour at room temperature with Blockin Buffer 2 comprising 5 % skim milk powder (Sigma Aldrich, Germany) in 0.05 % Polysorbat 20, 0.01 % skim milk powder in PBS pH 7.4 to block the free binding sites of the well surface. Subsequently the wells were incubated for two hours at room temperature with the recombinant human anti-IL6-antigen (R&D systems Inc., USA) at concentrations of 4 and ng/ml and for additional 1 hour with the biotinylated human anti-IL6 detection antibody (R&D systems Inc., USA). Afterwards the wells were incubated for 20 min at room temperature in the dark with Streptavidine-HRP conjugate (R&D systems Inc., USA). For the enzymatic reaction the TMB and H.sub.2O.sub.2 substrate solution (Thermo Fisher Scientific, Germany) was added and incubated for further 20 min at 37° C. After stopping the enzymatic conversion of 3, 3', 5, 5'-Tetramethylbenzidine TMB with 3.6 M H.sub.2SO.sub.4 leading to a color change from blue to yellow, the yellow product was detected photometrically at 450 nm using a plate reader. The procedure is schematically depicted in
[0152] Finally, the calculation of the reduction of surface adsorption based on the photometric absorption of the samples was performed as explained above.
[0153] Example 1.2: Reduction of the surface adsorption on polystyrene in the presence of liquid compositions comprising the positively charged amino acid arginine and arginine in combination with the non-polar; osmolytic amino acid glycine at different total amino acid concentrations.
[0154] In the first example, the surface adsorption in solutions comprising arginine and combinations of arginine with glycine at total amino acid concentrations of 72.3 mM, 100 mM, 361.42 mM and 500 mM in water was analysed as follows: [0155] Arg*HCl (500 mM) without other additives [0156] Arg*HCl (100 mM) without other additives [0157] Arg*HCl/Gly (500 mM): 130 mM Arg*HCl + 370 mM Gly (Argininge to Glycine ½.8) [0158] Arg*HCl/Gly (361.42 mM): 95 mM Arg*HCl + 266.42 mM Gly (Argininge to Glycine ½.8) [0159] Arg*HCl/Gly (100 mM): 29 mM Arg*HCl + 71 mM Gly (Argininge to Glycine ½.8) [0160] Arg*HCl/Gly (72.3 mM): 19 mM Arg*HCl + 53.3 mM Gly (Argininge to Glycine ½.8)
[0161] Surface adsorption was analysed in the anti-IL6-ELISA as described in example 1.1. As shown in
[0162] The addition of the non-polar, osmolytic amino acid glycine to the positively charged amino acid arginine in a molar excess and a concentration ratio of 2.8 : 1 (non-equimolar) resulted in a remarkable increase of reduction of the anti-IL6-antibody adsorption on the polystyrene medium binding plates to 60 % - 80 %. Even the combination of the positively charged amino acid arginine with the non-polar amino acid glycine in total amino acid concentrations of 72.3 mM and 361.42 mM smaller than the arginine concentrations of 100 and 500 mM and the concomitant reduction of the corresponding arginine concentration to 19 mM and 95 mM resulted in a similar reduction of the surface adsorption of the anti-IL6-antibody to the polystyrene medium binding plate.
[0163] The observed reduction in surface adsoption indiacates that the addition of a molar excess of glycine to smaller concentrations of arginine remarkably increased the amount of reduction of protein adsorption in comparison to higher molar concentrations of arginine alone.
[0164] Example 1.3: Reduction of the surface adsorption on polystyrene in the presence of liquid compositions comprising the positively charged amino acid arginine in combination with aromatic amino acids phenylalanine, tryptophan and histidine.
[0165] To exclude effects related to other buffer components in comparison to pure water used in the amino acid solutions in example 1. 2 amino acid solution in this example and the following examples were prepared in PBS at pH 7.4. Due to the limited solubility of aromatic amino acids, aromatic amino acids could not be used in equimolar concentration in relation to arginine in the respective solutions. The following solutions were analysed for surface adsorption in the anti-IL6-ELISA as described in example 1.1.: [0166] Arg*HCl (500 mM) without other additives [0167] Arg*HCl (100 mM) without other additives [0168] Arg*HCl/Phe (500 mM)= 375 mM Arg*HCl + 125 mM Phe [0169] Arg*HCl/Phe (100 mM)= 50 mM Arg*HCl + 50 mM Phe [0170] Arg*HCl/Trp (500 mM)= 465 mM Arg*HCl + 35 mM Trp [0171] Arg*HCl/Trp (100 mM)= 75 mM Arg*HCl + 25 mM Trp [0172] Arg*HCl/His (500 mM)= 325 mM Arg*HCl + 175 mM His [0173] Arg*HCl/His (100 mM)= 50 mM Arg*HCl + 50 mM His
[0174] As shown in Figure, a remarkably stronger reduction of the surface adsorption of the anti-IL6 coating antibody in the presence of the non-equimolar mixtures of the positively charged amino acid arginine and the aromatic amino acids in comparison to arginine alone was observed.
[0175] The addition of the aromatic amino acids to the molar concentration excess of the positively charged amino acid arginine resulted in the remarkable increase of the reduction of the % surface adsorption, particularly at the lower antigen concentration of 4 ng/mL (55.38 % arginine/phenylalanine; 55.18 and 57.77 % % arginine/tryptophan; 57.22 % arginine/histidine). In the case of the higher IL6-antigen concentration of 6 ng/mL the % reduction of the surface adsorption in the presence of equimolar concentrations of phenylalanine is comparable to arginine alone at a concentration of 100 mM (50.57 % arginine/phenylalanine versus 51.94 % arginine alone). Surprisingly, although arginine is present all mixtures at a molar excess at total amino acid concentration of 500 mM, the % reduction of the surface adsorption of the coating antibody is higher than in the presence of arginine alone at a concentration of 500 mM. For example, at the higher antigen concentration of 6 ng/mL the observed adsorption reduction was observed for arginine/phenylalanine at a total amino acid concentration of 500 mM to be 49.41 % versus 39.17 % arginine 500 mM alone. The addition of the aromatic amino acids tryptophan and histidine to arginine in a molar excess resulted in a stronger reduction of the surface adsorption compared to phenylalanine in combination with arginine in both concentration ranges (
[0176] In the presence of 100 mM of the positively charged amino acid arginine alone, the % reduction of the protein adsorption was around 43 - 52 %. In the presence of 500 mM arginine, the reduction of the surface adsorption of the anti-IL6-antibody was around 36 - 40 %. It should be noted, that the concentrations of the liquid compositions containing arginine alone were in each case higher than the arginine concentrations in the mixtures. So, the increasing effectivities of the liquid compositions containing a molar excess of arginine in combination with the aromatic amino acids against protein surface adsorption are clearly the result of the addition of the aromatic amino acids to the molar excess concentration of arginine.
[0177] Example 1.4: Reduction of the surface adsorption on polystyrene in the presence of liquid compositions comprising combinations of the aromatic amino acids phenylalanine, tryptophan and histidine with the non-polar amino acid glycine without arginine.
[0178] The following solutions amino acid solutions in PBS were analysed for surface adsorption in the anti-IL6-ELISA as described in example 1.1.: [0179] Arg*HCl (500 mM) without other additives [0180] Arg*HCl (100 mM) without other additives [0181] Phe/Gly (500 mM) = 125 mM Phe + 375 mM Gly [0182] Phe/Gly (100 mM) = 50 mM Phe + 50 mM Gly [0183] Trp/Gly (500 mM) = 35 mM Trp + 465 mM Gly [0184] Trp/Gly (100 mM) = 25 mM Trp + 75 mM Gly [0185] His/Gly (500 mM) = 175 mM His + 325 mM Gly [0186] His/Gly (100 mM) = 50 mM His + 50 mM Gly
[0187] As shown in
[0188]
[0189] Example 1.5: Reduction of the surface adsorption on polystyrene by a low ionic strength liquid composition comprising combinations of the aromatic amino acids phenylalanine, tryptophan and histidine and the polar amino acid serine.
[0190] To investigate the influence of high ionic strength on the protein adsorption induced by the dissolution of the excipients in the high ionic strength buffer PBS, the liquid compositions for this example were prepared by dissolution of the excipients in the low ionic strength buffer 10 mM sodium phosphate pH 7.4. Similar to Example 3 the liquid compositions were prepared in buffer in order to ensure the stability of the coating antibody during the 17 h incubation time at 2-8° C. during the coating step.
[0191] Amino acids were provided in equimolar concentration ratios in some samples, e.g. 50 mM: 50 mM in the case of the total amino acid concentration of 100 mM and 250 mM : 250 mM for the total amino acid concentration of 500 mM. Due to the limited solubility of the aromatic amino acids phenylalanine, tryptophan and histidine the mixtures with serine particularly for the high total amino acid concentration of 500 mM were prepared containing non-equimolar concentration ratios. In the case of the low molar concentration of 100 mM only the liquid composition comprised tryptophan and serine in non-equimolar concentrations. Accordingly, the liquid compositions comprised the polar non-charged amino acid serine in a molar excess concentration. The following solutions were prepared: [0192] Arg*HCl (500 mM) without other additives [0193] Arg*HCl (100 mM) without other additives [0194] Phe/Ser (500 mM) = 125 mM Phe + 375 mM Ser [0195] Phe/Ser (100 mM) = 50 mM Phe + 50 mM Ser [0196] Trp/Ser (500 mM) = 35 mM Trp + 465 mM Ser [0197] Trp/Ser (100 mM) = 25 mM Trp + 75 mM Ser [0198] His/Ser (500 mM) = 175 mM His + 325 mM Ser [0199] His/Ser (100 mM) = 50 mM His + 50 mM Ser
[0200] As shown in
[0201] The combination of aromatic amino acids with the polar amino acid serine seems to have a slightly lower efficacy in the reduction of surface adsorption of the anti-IL6 coating antibody in comparison to the non-polar glycine in combination with the aromatic amino acids (
[0202] Example 1.6: Reduction of the surface adsorption on polystyrene in the presence of liquid compositions comprising combinations of the aromatic amino acids phenylalanine, tryptophan and histidine and the negatively charged amino acids glutamic acid and aspartic acid.
[0203] As discussed in the previous examples, the addition of the non-polar osmolytic amino acid glycine and the polar non-charged amino acid serine in a molar excess over aromatic amino acids phenylalanine, tryptophan and histidine characterised by a limited water-solubility resulted in a remarkable reduction of protein adsorption on hydrophobic polystyrene medium binding plates.
[0204] To test other amino acid groups concerning the physico-chemical properties of the side chains combinations of the aromatic amino acids phenylalanine, tryptophan and histidine with the negatively charged amino acids glutamic acid and aspartic acid were analysed. Due to the limited water-solubility of both kinds of amino acids, the liquid compositions were prepared with remarkable lower total amino acid concentrations. Thus, the molar concentration ratios of the two amino acids were different to the liquid compositions tested in the above experiments and the total amino acid concentrations were significantly smaller compared to the arginine concentrations of 100 mM and 500 mM arginine. In the case of the higher total amino acid concentrations, the concentration of the aromatic amino acids were comparable to the liquid compositions according to the last example. The negatively charged amino acids were added in concentrations according to their maximal water-solubility. All liquid compositions were prepared in the low ionic strength buffer 10 mM sodium phosphate at pH 7.4 as follows: [0205] Trp/Glu (85 mM) = 35 mM Trp + 50 mM Glu [0206] Trp/Glu (42.5 mM) = 17.5 mM Trp + 25 mM Glu [0207] His/Glu (225 mM) = 175 mM His + 50 mM Glu [0208] His/Glu (112.5 mM) = 62.5 mM His + 50 mM Glu [0209] Phe/Glu (175 mM) = 125 mM Phe + 50 mM Glu [0210] Phe/Glu (87.5 mM) = 43.75 mM Phe + 43.75 mM Glu [0211] Trp/Asp (55 mM) = 35 mM Trp + 20 mM Asp [0212] Trp/Asp (27.5 mM) = 13.75 mM Trp + 13.75 mM Asp [0213] His/Asp (195 mM) = 175 mM His + 20 mM Asp [0214] His/Asp (97.5 mM) = 77.5 mM His + 20 mM Asp [0215] Phe/Asp (145 mM) = 125 mM Phe + 20 mM Asp [0216] Phe/Asp (72.5 mM) = 52.5 mM Phe + 20 mM Asp
[0217] Surprisingly, the addition of negatively charged amino acids to aromatic amino acids in liquid compositions at a lower total amino acid concentration compared to arginine alone with a molar concentration of 100 mM and 500 mM resulted in a comparable or even higher reduction of protein adsorption on the plate surface.
[0218] As shown in
[0219] Example 1.7: Reduction of the surface adsorption on polystyrene in the presence of liquid compositions comprising combinations of three amino acids.
[0220] In the next step, the effectivity in reducing protein adsorption of combinations of three different amino acids was analyzed by combining the liquid compositions according to the previous examples. Mixtures of aromatic amino acids with the polar non-charged amino acid serine and the negatively charged amino acid glutamic acid were prepared in 10 mM sodium phosphate buffer at pH 7.4. The total amino acid concentrations were adjusted to 100 mM and 500 mM in mainly equimolar concentration ratios with the exception for excipients with low solubility. In the case of high total amino acid concentration of 500 mM, the amino acid mixtures were not equimolar due to the limited solubilities of the aromatic amino acids and glutamic acid. Therefore, the polar amino acid serine was comprised in all liquid compositions with the highest total amino acid concentration as the amino acid with the molar excess. Amino acid mixtures with a total amino acid concentration of 100 mM were all equimolar. The applied liquid compositions contained the three amino acids were prepared with the following molar ratios: [0221] Arg*HCl (500 mM) without other additives [0222] Arg*HCl (100 mM) without other additives [0223] Phe/Ser/Glu (500 mM) = 125 mM Phe + 325 mM Ser + 50 mM Glu [0224] Phe/Ser/Glu (100 mM) = 33.33 mM Phe + 33.33 mM Ser + 33.33 Glu [0225] Trp/Ser/Glu (500 mM) = 35 mM Trp + 415 mM Ser + 50 mM Glu [0226] Trp/Ser/Glu (100 mM) = 33.33 mM Trp + 33.33 mM Ser + 33.33 mM Glu [0227] His/Ser/Glu (500 mM) = 175 mM His + 275 mM Ser + 50 mM Glu [0228] His/Ser/Glu (100 mM) = 33.33 mM His + 33.33 mM Ser + 33.33 mM Glu
[0229] As shown in
[0230] In order to increase the effectivity of the mixtures containing three different amino acids the polar uncharged amino acid serine was substituted by the non-polar small osmolytic amino acid glycine, leading to the combinations of aromatic amino acids with the non-polar osmolytic amino acid glycine and the negatively charged amino acid glutamic acid. The mixtures were prepared in the low ionic strength 10 mM sodium phosphate buffer at pH 7.4 similar to the previous compositions in this example. Total amino acid concentrations of 100 mM and 500 mM were prepared. Due to the limited solubility of the aromatic amino acids liquid compositions with a total amino acid concentration of 500 mM comprised non-equimolar mixtures of the three amino acids. Mixtures with a total amino acid concentration of 100 mM were prepared as equimolar amino acid mixtures. The non-polar hydrophobic amino acid was present in the 500 mM liquid compositions in a molar excess. The concentrations of the single amino acids are comparable to the mixtures of aromatic amino acids/serine/glutamic acid. The prepared liquid compositions comprised the following molar concentration ratios of the amino acids: [0231] Arg*HCl (500 mM) without other additives [0232] Arg*HCl (100 mM) without other additives [0233] Trp/Gly/Glu (500 mM) = 35 mM Trp + 415 mM Gly + 50 mM Glu [0234] Trp/Gly/Glu (100 mM) = 33.33 mM Trp + 33.33 mM + 33.33 mM Glu [0235] His/Gly/Glu (500 mM) = 175 mM His + 275 mM Gly + 50 mM Glu [0236] His/Gly/Glu (100 mM) = 33.33 mM His + 33.33 mM Gly + 33.33 mM Glu [0237] Phe/Gly/Glu (500 mM) = 125 mM Phe + 325 mM Gly + 50 mM Glu [0238] Phe/Gly/Glu (100 mM) = 33.33 mM Phe + 33.33 mM Gly + 33.33 mM Glu
[0239] As shown in
[0240] The addition of the non-polar osmolytic amino acid glycine to the mixtures of aromatic amino acids and the negatively charged amino acid glutamic acid revealed a remarkably increased ability of the liquid compositions to reduce protein surface adsorption (see Example 1.6;
[0241] Example 1.8: Reduction of the surface adsorption on polypropylen in the presence of liquid compositions comprising combinations of aromatic amino acids phenylalanine, tryptophane and histidine with the non-polar hydrophobic, branched amino acid leucine.
[0242] As many primary packaging systems for biological pharmaceutics, e.g. syringes, bags etc. are manufactured using polypropylene as material, we analyzed the effectivity of several amino acid mixtures against the adsorption of the anti-IL6 coating antibody on the hydrophobic surface polypropylene in addition to polystyrene plates used in experiments 1.2 to 1.7. In this example, combinations of aromatic amino acids phenylalanine, tryptophan and histidine with the hydrophobic non-polar branched chain amino acid leucine were analyzed. Due to the limited water-solubility of non-polar hydrophobic and aromatic amino acids, the liquid compositions were prepared with remarkable lower total amino acid concentration. Concentration ratios of the two amino acids were different compared to the previous examples and the total amino acid concentrations were smaller than 100 mM and 500 mM arginine. In the case of the higher molar concentrations, the molar concentration of the containing aromatic amino acids were comparable to the liquid compositions according to the previous example. The non-polar hydrophobic, branched amino acid was added in molar concentrations according to their maximal water-solubility. All liquid compositions were prepared in the low ionic strength buffer 10 mM sodium phosphate at pH 7.4 as follows: [0243] Trp/Leu (135 mM) = 35 mM Trp + 100 mM Leu [0244] Trp/Leu (67.5 mM) = 33.75 mM Trp + 33.75 mM Leu [0245] His/Leu (275 mM) = 175 mM His + 100 mM Leu [0246] His/Leu (137.5 mM) = 68.75 mM His + 68.75 mM Leu [0247] Phe/Leu (225 mM) = 125 mM Phe + 100 mM Leu [0248] Phe/Leu (112.5 mM) = 56.25 mM Phe + 56.25 mM Leu
[0249] As shown in
[0250] Example 1.9: Reduction of the surface adsorption on polypropylen in the presence of liquid compositions comprising combinations of aromatic amino acids phenylalanine, tryptophan and histidine with the negatively charged amino acids glutamic acid and aspartic acid.
[0251] Amino acid mixtures were prepared analogous to the similar mixtures according to Example 1.6. Due to the limited water-solubility of the amino acids used, the liquid compositions were prepared with remarkable lower total amino acid concentrations. Thus, the molar concentration ratios of the two amino acids were remarkably different to the previous liquid compositions and the total amino acid concentrations were remarkably smaller than the arginine concentrations of 100 mM and 500 mM. For the higher molar concentrations, the molar concentration of the comprised aromatic amino acids were comparable to the liquid compositions according to the previous example. The negatively charged amino acids were added in molar concentrations according to their maximal water-solubility. All liquid compositions were prepared in the low ionic strength buffer 10 mM sodium phosphate at pH 7.4 as follows: [0252] Trp/Glu (85 mM) = 35 mM Trp + 50 mM Glu [0253] Trp/Glu (42.5 mM) = 17.5 mM Trp + 25 mM Glu [0254] His/Glu (225 mM) = 175 mM His + 50 mM Glu [0255] His/Glu (112.5 mM) = 62.5 mM His + 50 mM Glu [0256] Phe/Glu (175 mM) = 125 mM Phe + 50 mM Glu [0257] Phe/Glu (87.5 mM) = 43.75 mM Phe + 43.75 mM Glu [0258] Trp/Asp (55 mM) = 35 mM Trp + 20 mM Asp [0259] Trp/Asp (27.5 mM) = 13.75 mM Trp + 13.75 mM Asp [0260] His/Asp (195 mM) = 175 mM His + 20 mM Asp [0261] His/Asp (97.5 mM) = 77.5 mM His + 20 mM Asp [0262] Phe/Asp (145 mM) = 125 mM Phe + 20 mM Asp [0263] Phe/Asp (72.5 mM) = 52.5 mM Phe + 20 mM Asp
[0264] As shown in