Expression lyophilization

Enzyme Reference Serums. Several companies sell lyophilized or stabilized reference serums for the calibration of instruments and for quality control. The label values given for the enzymatic activity of these serums should never be taken at face value, as at times they may be quite erroneous (19,33). Also, these values should only be used for the assay with which they were standardized, as interconversion of activity from one method to another for the same enzyme may often lead to marked errors. For instance, it is not recommended that alkaline phosphatase expressed in Bodansky units be multiplied by a factor to convert it to the units of the Ring-Armstrong method, or any other method for that matter. 

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

KNOB protein, however, was shown to improve gene expression markedly (130-fold in HeLa cells). Additionally, it was shown that PEG could be conjugated to the surface of the nanospheres to prevent aggregation during lyophilization without a loss of bioactivity following one month in storage. The PEGylated particles, however, were cleared from mice at a slower rate than unmodified controls and were found to accumulate in the kidney and liver at 15 min after intravenous administration. There was no difference after one hour, however. 

Although most assays perform well with regard to specificity and reproducibility, the major problem remains their standardization (A9, Dl, K30, L4). There is currently no internationally accepted standard, and the selection of a reference material raised many problems (A8, G5, K30, L4). A number of questions have not been solved Should the standard consist of several apo(a) isoforms Can the reference material be lyophilized Should results be expressed as mass or as moles of apoprotein or lipoprotein How should the protein mass of the primary standard be determined What are optimal storage conditions for the secondary standard Which method can be used as a reference method Can recombinant apo(a) represent an alternative for a primary standard These problems came to light in the course of the international surveys whose results were presented at the Lp(a) Workshop in New Orleans (1992) (L4). 

Dosage form BeneFix is formulated as a sterile nonpyrogenic, lyophilized preparation, intended for injection after reconstitution with sterile water for injection. It is available in single-use vials containing the labeled amount of factor IX activity, expressed in international units (lU). Each vial contains nominally 250,500, or 1000 lU of Coagulation Factor IX (Recombinant). 

Activase (Alteplase) [CHO-expressed glycoprotein-recombinant human tissue plasminogen activator] Genentech Lyophilized (50 mg single-dose vial 100 mg single dose vial) 50 and 100 mg doses to be reconstituted with 50 or lOOmL of WFI, respectively Per 50 mg vial 1.7 g L-arginine 0.5 g phosphoric acid < 4 mg polysorbate 80 (under vacuum) Per 100 mg vial 3.5 g L-arginine 1 g phosphoric acid <11 mg polysorbate 80 (without vacuum) IV... 

Beatseron (interferon-beta-lb) [E. coli expressed recombinant human interferon beta] Berlex, Inc. Lyophilized (0.3 mg single-dose vial)... 

Herceptin (trastuzumab) [CHO-expressed humanized recombinant IgGl monoclonal Ab] Genentech Lyophilized singledose vial... 

Humatrope (somatropin) [E. coli-expressed recombinant polypeptide hormone] Eli Lilly Lyophilized 5 mg single-dose vials... 

REFLUDAN (Lepirudin) [recombinant hirudin polypeptide expressed in yeast cells] Berlex Lyophilized 50 mg single-dose vial... 

SIMULECT (Basiliximab) [CHO-expressed glycoprotein, chimeric monoclonal Ab IgGl] Novartis Lyophilized 10 mg single-dose vial 20 mg single-dose vial... 

Optimization of the biotransformation showed that the best conditions for achieving high rates and molar conversions included the use of n-hexane and -heptane as solvents, a temperature of 50 °C, an equimolar concentration of the substrates around 50 mM, and a concentration of the lyophilized biocatalyst around 25-30 g/1. As a matter of fact, Table 6.1 shows how the carbon source affects the expressed activity of A. oryzae MIM and R. oryzae CBS 112.07. 

HIV recombinant-NCp7. HIV NC coding sequence was cloned into the pMal-c vector, expressed as a fusion protein in E.coli. After factor Xa cleavage, the 55 residues NC protein (Fig. 3) was purified by HPLC and complexed with two equivalent of Zn and stored as a lyophilized powder (NCp7). 

For QD and fluorescence spectroscopic analyses of (o-[l-Sei46]Aga-TK and co-[D-Ser46]Aga-TK, peptide samples were prepared by freshly dissolving the lyophilized peptides at a concentration of 150 ng/ml in Dulbecco s phosphate-buffered saline, pH 7.4, in the presence or absence of guanidine hydrochloride. CD spectra were recorded with a Jasco J-720WI spectropolarimeter at room temperature using a 0.1 cm path-length cell. In aU cases, the buffer base-line spectrum was subtracted, and the results were expressed in terms of the mean residue ellipticity... 

There is no general rule concerning the stability of a transferase e.g., / (1 — 4)GalT can be stored as a lyophilized powder and reconstituted in an appropriate buffer system before use. The transferases show optimum conversion rates at 37°C and at a pH around 7. Some enzymes like the GalTs and FucTs require Mn as a cofactor whereas SiaTs do not. The user should also pay attention to the different stabilities of the various transferases depending on the expression and purification protocols. 

After expression and purification, protein samples are often initially available as lyophilized solids, in which the proteins are correctly folded but in an amorphous arrangement with an isotropic distribution of orientations. This form of the protein may be used for NMR studies but the lack of water can significantly affect the strucmres of the individual protein molecules. One simple way to overcome this is to rehydrate the sample with small amounts of water." The effect can be quite significant, as illustrated in Fig. 2 for a sample of the 76-residue protein ubiquitin. Flash freezing offers the opportunity of preparing solid samples of proteins in which the solution-state structure is largely preserved." " ... 

The equilibrium number of particles of radius r per unit volume of a lyophilic colloidal system, n] (r), is given by the expression... 

The formation of this equilibrium colloidal system may take place if the value of d lies within the region where the particle size, d, is significantly larger than molecular dimensions, b d b, for instance in the region where d z (5 - 10)6. Then the necessary condition of spontaneous formation of lyophilic colloidal system, and, consequently, the condition under which this system may be in equilibrium with macroscopic phase, can be expressed by the Rehbinder - Shchukin criterion [3,4] ... 

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